attachment ..,, 5 comparative assessment of the...

Part D, Chdpter VII ’ Page 54 of 57 Mahyco B.t. cotton

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Comparative assessment of the allergenicity of cottonseed meal from BollgardTM and non-transgenic indian hybrid

cotton in a guinea pig model.

Monsanto Company Biotech Regulatory Science

Final Report

REPORT NO.: MSL-15’763

DATE: X2/5/98

TITLE: Comparative assessment of the allergenicity of cottonseed meal from BoJlgardTM and non-transgenic Indian hybrid cotton in a guinea pig model

AUTHOR: Richard E. Goodman

ABSTRACT: A study was conducted using guinea pigs to assess the relative allergenic activity of cottonseed meal from transgenic cotton, containing the CrylAc Lepidopteran specific insecticidal protein from Bacillus thuringiensis, compared to non-transgenic cotton.

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Young adult female Hartley-derived albino guinea pigs were fed 8s diets containing no cottonseed meal (control diet) or diets

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containing 10% (w/w> cottonseed meal made either from non- transgenic cottonseed (MECH-1 and MECH-3) or transgenic

E 8d a 2 -4 cottonseed (MECH-1 Bt) containing CrylAc. The diets were to be 8 pd fed for approximately 60 days and a passive ckaneous

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anaphylaxis assay (PCA) would be done to compare the antigen

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83 specific IgGla reaginic antibody response of all groups. However,

8g by day 21 guinea pigs fed cottonseed meal-containing diets gained 3 Sk significantly less weight compared to controls and failed to thrive. *r( ar

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3 ga As a result, all groups were fed commercial guinea pig diet from day 21 to day 28; guinea pigs subsequently were fed their

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intended diets from day 28 to day 49 at which time the study was

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The addition of cottonseed meal to the diet at 10% w/w produced an immediate decrease in body weight gain and lower weights when compared to controls. For example, by day 21 body weights of animals fed the non-transgen’k raw cottonseed meal diets, MECH-1 and MECH-3, were significantly less (p<O.OOl) than animals fed the control diet ( Table I). Weight gains in the MECH-1 and MECH-3 groups were on average only 22% compared to the control group. These animals also exhibited clinical signs including reduced fecal pellet production, soft stools and rough coats.

Distribution Copy Number

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1 D.Re BBlK 2 G. Fuller C2SB 3 P. Weston GG4F 4 R. Deaton BBIE 5 T. C. Lee BB5G 6 I. Lamb C2SE 7 K. Magin GG4K 8 R. Lirette BBSK 9 M. McKee C2SE 10 R Hileman BB5G 11 T. Fuhremann C2SE 12 P. Pyla BB5G 13. J. Lee BBSG 14. J. Leach BBSG 15. G. Holleschak BB5G 16. T. Nickson BB5F 17. G. Rogan , BB5K 18. P. Sanders BB5F 19. R. Sidhu BB5M 20. M. Taylor BB5F

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DO NOT RETURN IT TO THE LIBRARY

Abstract (continued) Table I

Mean Body Weights (g) of Guinea Pigs

Diets Day 0 Day 21 Day 28 Day 43 Control 333 444 484 551 Control I 332 443 473 523 MECH-1 Bt’ MECH-1 333 355 * i 405 * 443 * MECH-3 333 359 u 402 * 466 # ’ This group did not receive cottonseed meal in the diet from day 1 through day 28 and MECH-1 Bt from day 28-49. Significantly different from control: * pcO.001, # p<O.Ol.

On day 21, all animals were fed commercial diet for the next seven days to determine whether the animals would recover body weight. In addition, feed samples were analyzed for vitamin C, mycotoxins and CrylAc; results showed that the feed provided to the MECH-1 Bt group for the first 21 days did not contain CrylAc, subsequently it was determined that this group inadvertently had been fed control diet. Between days 21 and 28, MECH-1 and MECH-3 groups gained weight at’a rate similar to controls, except that one animal in the MECH-1 group died and one in the MECH-3 group was moribund and subsequently euthanized.

On day 28, remaining animals were fed their intended diets. Body weight gains were again decreased in cottonseed meal fed groups from days 29 to 35, whereas gains were lower or similar to control gains from days 36-49. Failure of the animals to thrive on 10% cottonseed meal indicated probatile compromised health status of these animals. The study was terminated without performing PCA assays on day 49.

In conclusion, results demonstrate that guinea pigs have decreased body weight gains and clinical signs (reduced fecal pellet size and abundance, soft stools and rough coat) when fed a nutritionally balanced diet containing 10% cottonseed meal (MECH-1 and MECH-3). Although a direct comparison of gains and signs between the MECH-1 Bt and MECH-1 and MECH-3 groups was not possible, the results show that guinea pigs fed diets with 10% (w/w> cottonseed meal&rave significant decreases in body weights and gains by day 21. Since weight gain for MECH-1 and MECH-3 groups were on average 22% of the weight gain of the control group during the first 21 days of this study, the likelihood that the animals would produce a reliable immune response was doubtful. Thus it was concluded that feeding guinea pigs 10% cottonseed meal for a 60 day sensitization period to accurately measure relative allergenic activity of the test diets was not possible.

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Final Report Monsanto Company Biotech Regulatory Sciences

Study #: 97-01-00-02 MSL-15763 Page #: 1 of 197

Studv Title

Comparative assessment of the allergenicity of cottonseed meal from BollgardTM and non-transgenic Indian hybrid cotton in a guinea pig

model

Sraonsor Andrew J. Reed

Author Richard E. Goodman

Study Report December &I998

Performinp Laboratories

Monsanto Company Biotech Regulatory Sciences

‘700 Chesterfield Parkway North St. Lo.uis, MO. 63198

Springborn Laboratories, Inc. 640 N. Elizabeth Street Spencerville, OW 45887

Laboratory Proiect ID 97-01-00-02


Study Number: 97-01-00-02

Study #: 97-01-00-02 M&-1576$ Page #: 2 of 197

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Title: Comparative assessment of the allergenicity of cottonseed meal from43011gardfM and non-transgenic Indian hybrid cotton in a guinea pig model

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Study Author : Richard E. Goodman -

Contributors: Larry R. Ho&den, Joan L. Lee, Gyula Holleschak, Terry Kaempfe, James D. A&wood -

Record Retention: All study specific raw data, protocols and final reports will be retained at Monsanto, St. Louis, except the raw data developed at Ralston Analytical Labs, for analysis of the raw cottonseed meal, which is retained at the Ralston Analytical Laboratory, 824 Gratiot, St. Louis, MO 63102. Actual dietary composition, other than the cottonseed, is proprietary and is held at the Richmond, IN facility of Purina TestDiet, 1050 Progress Dr., Richmond, IN 47374.

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Sample Retention: Any study samples which are to be retained will be stored at Monsanto, St. Louis. -

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Find Report Study #: 97~01-00-02

Monsanto Company MSL-15763 3iotech Regulatory Sciences Page #: 3 of 197

Signatures of Report Approval:

&udy Author Richard E. Goodman, Ph.D. Protein Characterization and Safety Center

Sponsor Andrew J. Reed, Ph.D. Biotech Regulatory Sciences

&%Glnager ’ ‘date James D. A&wood, Ph.D. Protein Characterization and Safety Center

Final Report Study #: 97-01-00-02

Monsanto Company MSL-15763

Biotech Regulatory Sciences Page #: 4 of 197

TABLE OF CONTENTS

Title Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Signatures of Final Report Approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1. Summary ............................................................................................ 7

2. Introduction, ..................................................................................... 8

2.1 Background ............................................................................... 9

2.2 Purpose .................................................................................... 10

3. Experimental Design .................................................................... 10

3.1 Materials ................................................................................. 10

3.2 Animals . . . . . . . . . . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

3.3 Feeding Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

3.4 Statistical Methods.. ...................................... .......................... 13

4. Results/Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . .,... . . . . . . . . . . . . . . . . . . . . . . . . ..a........ 13

4.1 Cottonseed Meal Analyses ..................................................... 13

4.2 Transgenic Protein Expression in Seeds ............................... 13

4.3 SDS-PAGE Band Patterns ..................................................... 13

4.4 Feed Manufacture ................................................................... 14

4.5 Feed Analysis Prior to Study Initiation ................................ 14

4.6 Animal Body Weights Days 0 to 21 ....................................... 15

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4.7 Immunoassay of CrylAc Protein in, Feed .............................. 17

4.8 Feed Analysis Post-Day 21 ..................................................... 17

4.9 Weight Gain Day 21 to Termination at Day 49 .................... 18

Conclusions/Discussion ................................................................ 19

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6. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..*.................. 21

7. Appendices . . ..a*......................*. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..I....... 22

7.1 Study Protocol ,.....,...,..............,.......,,..,,.,...,..,,.....,...........~...... 22

7.2 Springborn Final Report ........................................................ 27

7.3 Cottonseed Meal Analysis.. .................................................. 168

7.4 Feed Analysis Pre-Study ...................................................... 172

7.5 Feed Analysis Post-Day 21............................... .................... 185

Final. Report Study #: 97-01-00-02

Monsanto Company MSL-15763

Blotech Regulatory Sciences Page #: 6 of 197

CQdAC CrylAc

CSM DHL g mg NT ml d FcyR i.c. i.d. f&s MAHYCO MECH-1 MECH-3

ABBREVIATIONS

Class I (Lepidopteran-specific) crystal protein gene Class I (Lepidopteran-specific) crystal protein from BaciZZus thuringiensis var. kurstaki cottonseed meal DHL Worldwide Express gram milligram microgram milliliter microliter High affinity IgGla receptor intracardiac intradermal antibody isotype IgE Maharashtra Hybrids Seeds CO. Ltd. MAHYCO cotton hybrid #l MAHYCO cotton hybrid #3

MECH-1 Bt Transgenic MAHYCO cotton hybrid #l with CrylAc and NPTII nptI1 neomycin resistance, selectable marker gene (Neomycin

phosphotransferase II gene NPTII neomycin resistance protein (amino-glycoside-3’.

phosphotransferase II) SAS@ SAS@ statistical analysis software SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis SLI Springborn Laboratories, Inc. w/v weight to volume w/w weight to weight

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.Final Report Study #: 97-01-00-02~

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1. SUMMARY A study was conducted using guinea pigs to assess th,e relative allergenic activity of cottonseed meal from transgenic cotton, containing the CrylAc L’epidopteran specific insecticidal protein from BaciZZus thuringiensis, compared, to non-transgenic cotton. Young adult female Hartley-derived albino guinea pigs were fed diets containing no cottonseed meal (control diet) or diets containing 10% (w/w) cottonseed meal made either from non- transgenic cottonseed (MECH-1 and MECH-3) or transgenic cottonseed (MECH-1 Bt) containing CrylAc. The diets were to be fed for approximately 60 days and a passive cutaneous anaphylaxis assay (PCA) would be done to compare the antigen specific IgGla reaginic antibody response of all groups. However, by day 21 guinea pigs fed cottonseed meal-containing diets gained significantly less weight compared to controls and failed to thrive. As a result, all groups were fed commercial guinea pig diet from day 21 to day 28; guinea pigs subsequently were fed their intended diets from day 28 to day 49 at which time the study was terminated.

The addition of cottonseed meal to the diet at 10% w/w produced an immediate decrease in body weight gain and lower weights when compared to controls. For example, by day 21 body weights of animals fed the non- transgenic raw cottonseed meal diets (M&CH-1 and MECH-3) were significantly less (p<O.OOl) than animals fed the control diet (Table I). , Weight gains in the MECH-1 and MECH-3 groups were on average only 22% compared to the control group. These animals also exhibited clinical signs including reduced fecal pellet production, soft stools and rough coats.

’ Table I Mean Body Weights (g) of Guinea Pigs

Diets Day 0 Day 21 Day 28 Day 49 Control 333 444 484 551 Control / 332 443 473 529 MECH-1 Bt’ MECH-1 333 35SZh ’ 405 * _ 443 * MECH-3 333

I 359 * 402 * 466 #

’ This group did not receive cottonseed meal in the diet from day 1 through day 28 and MECH-1 Bt from day 28-49. Significantly different from control: * pcO.001, # pcO.01.

On day 21, all animals were fed commercial diet for the next seven days to determine whether the animals would recover body weight. In addition, feed

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Final Report Study #: 97-01.00-02 Monsanto Company MSL-16763 Biotech Regulatory Sciences Page #: 8 of 197

samples were analyzed for vitamin C, mycotoxins and CrylAc; results showed that the feed provided to the MECH-1 Bt group for the first 21 days did not contain CrylAc, subsequently it was determined that this group inadvertently had been fed control diet. Between days 21 and 28, MECH-1 and MECH-3 groups gained weight at a rate similar to controls, except that one animal in the MECH-1 group died and one in the MECH-3 group was moribund and subsequently euthanized.

On day 28, remaining animals were fed their intended diets. Body weight gains were again decreased in cottonseed meal fed groups from days 29 to 35, whereas gains were lower or similar to control gains from days 36-49. Failure of the animals to thrive on 10% cottonseed meal indicated probable compromised health status of these animals: The study was ‘terminated without performing PCA assays on day 49.

In conclusion, results demonstrate that guinea pigs have decreased body weight gains and clinical signs (reduced fecal pellet size and abundance, soft stools and rough coat) when fed a nutritionally balanced diet containing 10% cottonseed meal (MECH-1 and MECH-3). Although a direct comparison of gains and signs between the MECH-1 Bt and MECH-,l and MECH-3 groups was not possible, the results show that guinea pigs fed diets with 10% (w/w) cottonseed meal have significant decreases in body weights and gains by day 21. Since weight gain for MECH-1 and MECH-3 groups were on average 22% of the weight gain of the control group during the first 21 days of this study, the likelihood that the animals would produce a reliable immune response was doubtful. Thus it was concluded that feeding guinea pigs 10% cottonseed meal for a 60 day sensitization period to accurately measure relative allergenic activity of the test diets was not possible.

2. INTRODUCTION BollgardTM (a registered trademark of Monsanto Co.) cotton contains the crylAc gene encoding the CrylAc Lepidopteran specific insecticidal protein that was originally isolated from Bacillus thuringiensis var. kurstaki. The crylAc gene was transferred into cotton along with the marker gene, nptII, which expresses the neomycin phosphotransfer&e II (NPTII) protein. Monsanto Company and Maharashtra Hybrids Seeds CO. Ltd. (MAHYCO) have crossed Bollgard cotton with Indian cotton (MECH-1) to incorporate the crylAc gene into Indian cotton hybrids to provide protection against Lepidopteran insect pests in India.

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2.1 Background

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Various Bacillus thuringiensis formulations have been safely used for more + than thirty years to control insect pests in a variety of crops, ornamentals and trees (reviewed in Entwistle et al., 1993). As literature searches indicate; this use has not led to any documented cases .of allergic responses to the insectioidal proteins. CrylAc is insecticidal only to lepidopteran larvae when ingested. ‘CryZAc acts by binding to a lepidopteran larval specific mid- gut membrane receptor and after activation in the highly basic gut fluid, inducing perforation of the gut. CrylAc has no adverse biological effect on non-target species including mammals. More detailed background information is presented in MSL-15740 (Goodman and Holden, 1998). Searches of the literature and studies that we have performed (Astwood, 1995a; Ream, 1994; Ream, 1993; Fuchs et al., 1993; Astwood, 1995b) indicate that there is no evidence that would implicate either CrylAc or NPTII proteins as allergens. In addition, there is no evidence that any of the transgenic cotton materials are more allergenic than non-transgenic cotton.

The current study was performed in response to the request from the Indian Department of Biotechnology to further assess in a guinea pig model, the relative allergenic activity of transgenic Indian Bollgard cottonseed compared to non-transgenic Indian cottonseed. The basis for the assay is that Hartley guinea pigs develop reaginic antibodies, predominantly IgGla, in response to a wide range of antigens (Sarlo et al., 1994, Zhou et al., 1998). Antigen’. specific antibody concentrations or titers may be measured by passive cutaneous anaphylaxis (PCA). Since the primary concern for increasing the allergenicity of agricultural products is the risk associated with consumption, the logical route of sensitization is including the test material in the diet of nai’ve guinea pigs (Pahud and Schwarz, 1984). However, no animal model has been validated and shown to be an accurate predictor of the allergenic activity of a range of clinically relevant allergens (Metcalfe et al., 1996).

Guinea pigs do not normally consume cottonseed or cottonseed meal. There are no known reports establishing safe levels of consumption of cottonseed meal in this species. A primary concern regarding the use of any animal species to test cottonseed meal is the ability t&o&rate gossypol. Gossypol is a polyphenolic compound that is known to be more toxic to monogastric species than to ruminants (Abraham and Hron, 1992). There is little data concerning the safety levels for guinea pigs, but the acute L&0 may be as low as 280 mg/kg, nearly ten fold lower than the level that is safe for rats (Abou- Donia, 1989). If that number is valid, a 400 g guinea pig would be expected to consume around 20 g of feed per day, or 2 g of cottonseed meal. Assuming

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1% of the cottonseed weight is gossypol (Nida et al., 1996), the animal would consume 20 mg of gossypol, or approximately 12 to 15% of the LD50 dose, on a daily basis for 60 days. It is not clear what effect that level of consumption would have on the animals. Based on reports in the literature for domestic pigs, it is reasonable to assume that there could be serious health effects in guinea pigs due to chronic ingestion of cottonseed. Therefore, one objective of these studies was to assess whether guinea pigs would tolerate extended feeding of raw, full-fat cottonseed meal (CSM) at 10% of the diet.

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2.2 Purpose The purpose of this study was to assess the relative allergenicity of Indian Bollgard cottonseed meal compared to conventional cottonseed meal using a PCA assay by comparing the reaginic antibody response of Hartley guinea pigs fed either transgenic or non-transgenic cottonseed meal.

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3. EXPERIMENTAL DESIGN Prior to the initiation of this study, a similar study using the Brown Norway rat as a test animal was initiated under Study Protocol # 98-01-36-01. The overall design of the current study (protocol amendment request submitted to the Dept. of Biotechnology by Dr. Muzumdar, Appendix 7.1) was to sensitize Hartley guinea pigs to cottonseed proteins by feeding different groups of animals cottonseed meal from transgenic (MECH-1 Bt) and non-transgenic (MECH-1 and MECH-3) hybrid cotton. A non-cottonseed diet was included as an additional control. Serum from all animals would then be tested for the production of cotton specific IgGla by the in uivo method of PCA. Ten animals were assigned to each feeding group, non-cottonseed control, and 10% cottonseed meal diets of MECH-1 Bt, MECH-1 (non-transgenic) and MECH-3 (non-transgenic). At the end of the study, serum was to be collected from each animal, and used in a cross-over PCA assay designed to compare the relative reactivity of serum from each sensitization group and similarly dosed challenge groups. Skin reaction sizes were to be scored for comparison between hybrid extracts, doses and diets. This design allows direct comparison of each cottonseed test extract material while reducing the influence of animal to animal variation.

3.1 Materials a. Cottonseed. Seed of transgenic cotton (MECH-1 Bt) and non-transgenic cotton (MECH-1, MECH-3) were harvested, acid delinted by MAHYCO and shipped via DHL, through U.S. Customs to Monsanto Company. .

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b. Raw cottonseed meal (CSM). Cold (-200C) delinted cottonseed lots were weighed and ground to a moderately fine powder in the presence of liquid nitrogen, using a commercial/industrial Waring blender,. Powdered meals were stared frozen (-200(Z), with permeable covers and mixed occasionally over one day to allow the Nz to sublimate from the meal. The ground cottonseed meals’were maintained frozen (-200C). Proximate analysis was conducted on ground, whole cottonseed meal of all three hybrids. Raw, ground seed of the three hybrids was used in the manufacture of the feed for the guinea pigs.

c. Defatted cottonseed meal. Samples of raw CSM of each of the three hybrids were defatted by three serial extractions of each ground meal in six volumes of hexane (HPLC, W spectrophotometry grade) at 450d to 500C with mixing for 15 minutes. The hexane was removed by filtration after each extraction. Residual hexane was removed by evaporation at room temperature under vacuum. Defatted samples were used for protein gel electrophoresis, for immunoassay of CrylAc in the CSM and for production of challenge doses for the passive cutaneous anaphylaxis (PCA) assays.

d. Cottonseed meal analysis. Samples of raw cottonseed meal (not defatted) were submitted to Ralston Analytical Labs for analysis of gossypol content and for .proximate analysis.

e. Guinea pig diet pellet formulation. Dr. Dorrance Haught, primary animal nutritionist of Purina TestDiet in St. Louis, MQ, formulated three diets based on standard, commercial Purina Laboratory Guinea Pig Diet 5025. These were a control diet containing no cottonseed meal and three feed sensitization diets containing full-fat, ground, raw, transgenic MECH-1 Bt, non-transgenic MECH-1 and non-transgenic MECH-3 cottonseed. The percentage of cottonseed in the cottonseed containing diets was equal to 10% of the bulk weight of the diet. All four diets were balanced with approximately equal composition of total protein, lipid, carbohydrate and fiber. The other major plant material sources were soybeans, corn, oats, sugar beet, wheat and alfalfa. The diets contained fish meal and domestic animal meat. The exact proportion of components in&e diets is proprietary (Purina) except for the concentration of cottonseed meal.

f. Guinea pig diet pellet manufacture. Four test diets were manufactured at Purina TestDiet, Richmond, IN. Each diet was manufactured as two individual batches, dried and packed into three containers (boxes) for

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shipment to Springborn laboratories. Retention samples were,submitted to Monsanto.

g. Cry& detection assay. A qualitative immuno-detection assay was used to measure CrylAc protein in seed samples and dietary pellets using a CrylAc specific inonoclonal antibody and detection system. This assay was used to demonstrate that the MECH-1 Bt test materials contained the transgenic protein and that non-transgenic materials did not.

3.2 Animals Guinea pigs. Nulliparous, non-pregnant female Hartley guinea pigs between 300 g total body weight (approximately 5 to 7 weeks of age) were purchased from Charles Rivers Laboratories, Raleigh, NC by Springborn Laboratories. Animals were acclimated on control diet (Purina 5026) with food and deionized water provided ad libitum. All animals were marked with uniquely numbered ear tags. Animals were stratified by body weight and assigned to the feeding groups to minimize average weight differences.

3.3 Feeding Exposure Springborn Laboratories Inc., of Spencerville, OH conducted these studies. Protocols and results are included in the Final Report from Springborn (SLI Study # 3044.6971, Appendix 7.2. Following,acclimation, the animals were fed from box #2 of each of the appropriate dietary boxes for the first 21 days of the study (July 24, 1998 to August 14, 1998). As will be discussed in the results, on day 21 it became evident that the two non-transgenic CSM diets were not being consumed at a rate that would maintain the health status of the animals. All animals of the study were placed on commercial guinea pig diet for the next seven days to allow recovery of the health status of the animals, and to provide an opportunity to perform analyses of all diets. At day 28 of the study analytical results were equivocal, but most of the animals were gaining weight at a rate equal to the control diet growth rate. At day 28 all animals were returned to the scheduled dietary treatment, however feed was supplied from box #l of each diet instead of box #2; In addition, on day 31, all animals were placed on water bottles with supplemented vitamin C to help ensure sufficient vitamin C intake for thos~nimals that were not consuming adequate diet. Within one week after re-starting the treatment diets, all three CSM dietary groups were gaining significantly less weight than the control diet group and the MECH-1 Bt group lost an average of 5% body weight. On day 49 the study was terminated and all animals were euthanized due to the questionable health status and potential confounding effect on that might have on the immune system and study results.

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3.4 Statistical Methods Analysis of body weight data was performed at Monsanto using SASS (SAS Institute Inc., Cary, NC), by a one-way analysis of variance. Springborn Laboratories, Inc., performed separate data analyses which are presented in the final report,.Appendix 7.2.

4. RESULTS/OBSERVATIONS 4.1 Cottonseed Meal Analyses Cottonseeds were ground in liquid nitrogen (N2) at Monsanto in preparation for feed, analytical work and for the passive cutaneous anaphylaxis assay. Samples of the ground meal were transferred by courier to Ralston Analytical Labs, St. Louis, MO for analysis. Data from the individual analyses (Appendix 7.3) are summarized in Table II.

Table II. Summary data of Ralston Analytical proximate analysis of cottonseed meal samples, reported as percents of meal.

Cottonseed meal 1 MECH-1 Bt MECH-1 MECH-3 I

Moisture % 8.36 7.73 8.76 Protein % l 23.0 23.1 23.6 Ammonia % 0.14 0.13 0.14 Fat % 2 13.8 14.5 14.3 ’ Crude Fiber % 19.4 19.5 I 18.0 Ash% 4.28 3.92 4.21 Gossypol % 3 0.48 0.52 0.40 1 Protein measured by the protein combustion-IR method; 2 Fat determined by soxhlet extraction with ether; 3 free gossypol determined by HPLC

4.2 Transgenic Protein Expression in Seeds Seed samples from the cottonseed hybrids provided by MAHYCO were ground and extracted in neutral buffer, then tested using a CrylAc specific solid phased immunoassay. The MECH-1 Bt seed sample was the only that tested positive for the CrylAc protein.

4.3 SDS-PAGE Band Patterns The banding patterns of the CSM extracts in colloidal blue stained gel indicated that there were no obvious differences in the extracted proteins of the three CSM preparations (MECH-1 Bt, MECH-1 or MECH-3).

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4.4 Feed Manufacture Purina TestDiet manufactured the guinea pig feeding pellets based on the Purina 5025 guinea pig diet. A control diet was made using the same components as the test article diets, but without CSM. To manufacture the CSM diets, portions of components of the proprietary feed were replaced with raw, full-fat kottonseed meal such that 10% of the bulk of the diet was from either MECH-1 Bt, MECH-1 or MECH-3. The ingredient mix of the other components were adjusted to provide approximately equal levels of protein, lipid, carbohydrate and fiber in each diet, as summarized in Table III. The exact formulation was determined by the Purina TestDiet Nutritionist, based on the proximate data for the cottonseed meal provided by the Ralston Analytical Labs assay. Historical data of composition of the individual ingredients were used to formulate the diets. Contents included corn, oats, alfalfa, sugar beet pulp, wheat germ, wheat middlings, soybean meal, fish meal, meat meal, brewer’s yeast, micro-nutrients, calcium carbonate, sodium chloride, whey, DL-methionine, tallow, molasses, choline chloride and water. Diets were made sequentially, with the last completed on June 5, 1998, shipped directly to Springborn Laboratories. Retention samples were shipped to Monsanto.

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Table III. Calculated dietary composition, percent of total mass, calculated values’estimated from proximate analysis of cottonseed meal and historical data of other components.

1 Controi 10% MECH-1 Bt2 10% MECH-12 10% MECH-32 Protein1 I 19.3 20.1 20.1 20.1 Fat (SoxhletY 4.3 4.3 4.3 4.3 Crude Fiber’ 13.0 13.8 13.8 I 13.7 Starch1 17.3 12.8 12.8 12.8

,Ashl 8.1 7.6 7.6 I 7.6 1 percent by weight; 2 cottonseed meal, raw, full-fat

4.5 Feed Analysis Prior to Study Initiation Samples of all diets were submitted to Ralston Analytical Laboratories. Proximate analysis, iron, vitamin C and free gossypol determinations were performed on samples from boxes #1 and #2 of each diet. Pesticide residue analyses were performed on samples from box #2 $ each diet. Diets were manufactured approximately one month prior to-sampling. Values for each box of feed used in this study were determined (Table IV). Two values were lower than the other diets. Although low, vitamin C at 754 ppm in box#l of MECH-3 was still approximately three times greater than the minimum required for guinea pigs. The protein concentration of the sample from box #2 of MECH-3 (13.8%) was lower than other samples. New samples were

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submitted and analyzed in duplicate (17.2% and 17.4%), indicating that the protein concentration in this diet was sufficient. The concentration of free gossypol in all diets was low and was not expected to cause toxic effects. Values for pesticide residues were below the limits of detection:

Table IV. Measured analytical values one month aRer manufacture. Samples were analyzed by Ralston Analytical Laboratories.

Box #1 of each3 Control 10% MECH-1 Bt? 10% MECH-1” 10% MECH-32 Moisture 10.8 8.00 8.98 13.0 Protein1 19.9 20.6 20.1 20.1 Fat (SoxhleW 4.78 5.25 5.46 4.63 Crude Fiber1 13.6 16.2 15.7 14.5 Ash1 7.12 7.71 7.41 ’ 7.4 Iron (ppm) 281 298 280 276 Vitamin C (ppm) 1070 1200 1100 754 free gossypoll 0.011 0.034 0.037 0036

Box #2 of each4 Control 10% MECH-1 Bt2 10% MECH-12 10% MECH-32 Moisture 7.72 10.4 11.6 12.2 Protein’ 20.4 19.4 19.6 E19.61’ 13.8*[ 17.337 Fat (SoxhleW I 4.79 4.56 5.66 5.44 Crude Fiber1 14.0 13.6 14.5 17.8 Ash1 7.68 7.96 7.77 8.08 Iron (ppm> 275 I 280 259 280 Vitamin C (ppm) 1320 (1250 1230 I 1220’ free gossypoll 0.01 0.0115 0.041 I 0.04 carbamatess nd nd nd nd org.phosphates6 nd nd nd nd chlorinated pest.6 nd nd nd nd

1 percent by weight; 2 cottonseed meal, raw, full-f&; 3 samples fkoni Box#l of each diet (stored at room temperature for 30 days prior to sampling; J sampies from Box#2 of each diet, (stored refrigerated for 30 days prior to sampling); 5 note that this low free gossypol value measured for this box of feed is one of the few measurements that clearly supports the idea that box#2 of the MECHl Bt diet did not contain cottonseed; nd, not. detected; 6 broad spectrum pesticide screen, all values below the limit of detection. * Out of range protein value. 7 New sample analysis for protein only, 12/3/98 by Ralston Analytical.

4.6 Animal Body Weights Days 0 to 21 Each animal was weighed approximately weekldA;pendix 7.2). Cumulative mean percent weight gains over the 21 day feeding period (Figure 1) were compared statistically using a one-way analysis of variance. There were significant differences among weight gains between diet groups, establishing that cottonseed diets had an adverse impact oil growth rates. On day 21, the

* Springborn Study Director notified Monsanto of the weight gain discrepancy

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Biotech Regulatory Sciences Page #: 16 of 197

and early clinical observations that suggested there was a significant depression of the guinea pig growth rates with two of the cottonseed diets. Samples of dietary pellets from each box of feed were taken by Springborn and shipped to Ralston Analytical for vitamin C and mycotoxin assays. Similar samples were submitted to Monsanto for immunoassay detection of the CrylAc protein. On day 21 the test diet feed was removed from all cages and commercial diet (.3/ ,urina 5025) was supplied ad Zibitum for the next seven days. During the seven day recovery period the animals were monitored closely. One animal from the MECH-1 group died and one animal from the MECH-3 group was euthanized because it failed to gain weight and clinical symptoms indicated that the animal was moribund. All other animals gained weight at rates approximately equal to the control group and appeared to have recovered sufficiently by day 28 to restart the study. I

Guinea Pig Average Percent Body Weight Gain at 21 Days

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Figure 1. Average percent body weight gain at day 21 of the study. Treatment averages were compared to the control dietary group. This was the tist indication that guinea pigs would not gain weight normally on 10% full-fat cottonseed meal diets. Analysis of all boxes of diet following this finding demonstrated that the 10% MECH-1 Bt group in fact had been

’ fed control diet (without cottonseed) for the first 21 days of the study and had not received the intended cottonseed diet. No other differences were found.

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4.7 Immunoassay of CrylAc Protein in Feed Samples of each box of feed (3 per diet), returned by Springborn Laboratories for identity analysis were ground and extracted, and the supernatants were tested for the presence of the CrylAc protein. The results were as expected except that box #3 of the Control diet tested positive for CrylAc, and box #2 of the MECH-1 Bt diet tested negative for CrylAc. Subsequent testing of retention samples at Monsanto, and directly on-site at Springborn Laboratories, verified these results and indicated that the error occurred during packaging of those two boxes of diet. The results established that the MECH-1 Bt feeding group had not received CSM diet during the first 21 days of the study, but instead had received the non-cottonseed control diet, and therefore were equal to the control diet fed group during the initial feeding. When feeding of test diets was resumed at day 28, all animals were fed from box #l of the specified diets, which tests had shown were correct regarding the presence of the transgenic cottonseed meal.

4.8 Feed Analysis Post-Day 21 Because of the lack of weight gain in the non-transgenic cottonseed fed groups; samples of all three boxes of each diet (12 in all) were sent to Ralston Laboratories for analysis of vitamin C and 19 mycotoxins. Animals were only fed from boxes #l and #2 during this study. Lack of vitamin C, or the hresence of mycotoxins were potential causes of the compromised health of the animals. Some of the mycotoxin assays were performed by Romer Laboratories, on a subcontract from Ralston. Vitamin C values were all above 1,000 ppm except box #1 of the MECH-3 diet which at 793 ppm was similar to the previous determination of 753 ppm, and well above the minimum required dietary requirement. Only one mycotoxin was measured above the level of detection. That was deoxynivalenol (vomitoxin), which was measured in all dietary boxes including control diets. Fusarium infected corn was the most likely source of this mycotoxin. The measurements of deoxynivalenol were between 0.2 and 0.3 ppm and were not considered significant as the animals fed the control diet and the MECH-1 Bt diet (box #2) had the same level of the toxin and showed no ill effects. The nutritionist at Purina was consulted along with Monsanto veterinary pathologists and there was a general agreement that the levels ooeoxynivalenol were insignificant. The conclusion from this analysis was that the low weight gain, and apparent unpalatability of the cottonseed diets were not caused by a loss of vitamin C, and were not likely to have been caused by mycotoxins. Since the animals seemed to have recovered by day 28 on commercial diet and the specific cause of the weight gain reduction in CSM diets hadn’t been

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identified, a decision was made to restart the study using box#l from each diet, which were demonstrated to contain the correct test articles.

4.9 Weight Gain Day 21 to Termination at Day 49 Following the seven day recovery period (day 21 to day 28) when animals were on commercial diet, the correct treatment diets were provided ad Zibitum from box #1 of each diet on day 28. Additional vitamin C was provided in drinking water at day 31 since some of the earlier clinical symptoms suggested that some of the animals had symptoms consistent with scurvy which would be attributable to inadequate dietary intake of vitamin C. On day 35 at the next body weight measurement, all three CSM feeding groups were either loosing weight, or gaining weight at a significantly lower rate than the control group. This finding indicated that all three cottonseed diets were compromised. Although the rate of weight gain increased from day 35 to day 42, the total body weight differences between the control animals (averaging around 550 g at day 42) and the two non-transgenic cottonseed groups (averaging around 450 g at day 42) indicated that the health of the cottonseed fed groups was impaired. The study was stopped and all animals were euthanized on day 49. Because of the compromised health status of the animals, the planned immunological assays (??A) were not performed. The body weights were measured on the day of termination, day 49, and analysis indicated that although the animals were gaining weight, the animals were substantially smaller than the control animals (Figure 2).

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Mean Body Weights(g)

I~+MKHiBT -m-CmtroUME?+l Et -h-MfD+l --+-MEW-3 -a-.Cmtrol

0 7 14

Day of

Figure 2. Mean body weights. Animals were weighed at approximately weekly intervals. Average group weights are shown. While the average weights were equal initially, at the time of termination there was approximately a 100 g difference in total body weight between non-transgenic cottonseed fed animals and controls control fed animals. The weight difference indicates a highly likelihood of significant differences in the health and immune status of the animals. AU animals were euthanized on day 49 of the study and the sera was discarded. MECH-1 Bt group were unintentionally fed control diet during the first 21 days of the study and are indicated as MECH-1 Bt(CTL).

The primary conclusion of this study was that guinea pigs do not thrive on a diet containing 10% raw, full-fat cottonseed mealfor an extended period of time: Assay of vitamin C levels and tests foFa broad spectrum of mycotoxins failed to provide an answer regarding the cause of the unsuitable response to the cottonseed meal diets. There was evidence that the depressed weight gain was due to feed refusal. Animals fed the cottonseed meal diet gained less weight than controls, but recovered the rate of gain in seven days. However, reintroduction of the cottonseed meal diets at day 28 caused weight loss in two groups and little gain in the other. In addition, observations

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indicated that some, of the cottonseed meal fed animals were spilling feed, without evident consumption. As a consequence of the failure to thrive on these diets, the guinea pig model could not be used to accurately measure the relative allergenic activity of the transgenic and non-transgenic cotton hybrids.

Alternatively the Brown Norway rat model could be used to assess relative allergenicity of transgenic and non-transgenic cottonseed meal. These animals have been demonstrated to be tolerant to feeding 10% raw, full-fat cottonseed meal in t,he diet for sixty days (Goodman and Holden, 1998). The Brown Norway rat model has been used to evaluate potential changes in endogenous cottonseed allergens in transgenic hybrids relative to conventional cotton. No differences were found in the rat study using the same hybrids tested in this guinea pig study (Goodman and Holden, 1998).

6. REFERENCES

Abraham, G., and Hron R.J. Cilseeds and their oils. Hui, Y.H. ed. Encyclopedia of food science and technology, vol. 3., pp.1901-1910. 1992. John Wiley & Sons, Inc. New York, NY.

Abou-Donia, M.B. in Toxicants of plant origin, vol. IV, Phenolics. P.R. Cheeke ed. ’ 1989. CRC Press, Inc., Boca Raton, FL.

A&wood, J.D. Bacillus thuringiensis subsp. hurstahi, HD-73 insecticidal protein (B.t.k. HD- 73 protein) shares no significant sequence similarity with proteins associated with allergy or coeliac disease. 1995a., Monsanto Technical Report, MSL-14173, St. Louis, MO.

Astwood, J.D. Neomycin phosphotransferase II (NPTII) shares no significant sequence similarity with proteins associated with allergy or coeliac disease. 1995b. Monsanto Technical Report, MSL-14177, St. Louis, MO. ”

Entwistle, P.F., Cory, J.S., Bailey, M.J. and Higgs, S. (eds). Bacillus thuringiensis, an environmental biopesticide: Theory and practice. 1993. John Wiley & Sons. Chichester, England.

Fuchs, R.L., Ream, J.E., Hammond, B.G., Naylor, M.W., Leimgruber, R.M., and Berberich, S.A. Safety assessment of the neomycin phosphotransferase II (NPTII) protein. 1993. BiolTechnology 11:1543-1547.

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Goodman, R.E. and Holden, L.R. Assessment of the allergenicity of BollgardTh’ cottonseed proteins relative to conventional cottonseed proteins. 1998. Monsanto Technical Report, MSL-15740. St. Louis, MO.

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Metcalfe, D.D., A&wood, J.D., Townsend, R., Sampson, H.A., Taylor, S., andFuchs, R.L. Assessment of the allergenic potential of foods derived from genetically engineered crop plants. 1996. Crit, Rev. Food Sci. Nutr. 36(S):S165-S186.

Nida, D.L., Patzer, S., Harvey, P., Stipanovic, R., Wood, R. and Fuchs, R.L. Glyphosate- tolerant cotton: The composition of the cottonseed is equivalent to that of conventional cottonseed. i996. J. Agric. Food Chem. 44:1967-1974.

Pahud, J.J. and Schwarz, K. Research and development of infant formulae with reduced allergenic properties. 1984. Ann. Allergy “53(6 Pt 2):609-614.

Ream, J.E. Assessment of degradation of Neomycin phosphotransferase II in in vitro mammalian digestion models. 1993, Monsanto Technical Report, MSL-12290, St. Louis, MO.

Ream, J.E. Assessment of the in vitro digestive fate of Bacillus thuringiensis var. kurstaki HD-73 protein. 1994. Monsanto Technical Report, MSL-13299, St. Louis, MO.

Sarlo, K., Clark, E.D., Ferguson, J., Zeiss, C.R. and Hatoum, N. Induction of type I hypersenitivity in guinea pigs after inhalation of phthalic anhydride. 1994. 5. Allergy Clin. Immunol. 94:74’7-756.

Zhou D., Chen, G., Kim, J.-T., Lee, L.-Y. and Kang, B.C. A dose-response relationship ) between exposure to cookroach allergens and induction of sensitization in an experimental

asthma in Hartley guinea pigs. 1998. 3. Allergy Clin. Immunol. 101:653-659.

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APPENDICES: Assessment of the allergenicity of Bollgard cottonseed proteins relative to conventional cottonseed proteins.

Appendix 7.1 Study Protocol

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Animal Model Allergenicity Testing, Transformed Cotton

Amendment Request (4/17/98) for:

IV. PROTOCOL FOR ALLERGENICITY TESTING OF GENETICALLY TRANSFORMED PRODUCTS IN ANIMAL MODEL.

Submitted by Dr. P. Muzumdar, to The Department of Biotechnology, Government of India, New Dehli.


Animal Model. Unfortunately, no established animal model is available in the literature for assessing the allergenicity of genetically modified foods,.however, rabbit or guinea pig could be used to detect allergenicity. Brown Norway rats (Atkinson and Miller, 1994; Atkinson, et al., 1996) have been used in experimental studies but it is yet to be accepted as an animal model by the regulatory agencies. However, this could be used-to generate additional d&a.

Treatment Schedule: Normal adult healthy animals are kept under proper husbandry conditions with 12 hour light and 12 hour dark periods. Before the start of the experiment, animals are kept at least five days for acclimation. Animals are randomly divided into four groups, based on diet fed. The experimental group consists of ten animals, sensitized to the test protein(s) by incorporating the transgenic food stuff (crop material, variety or line) into the feeding pellets such that 10% of the total diet is from the test compound. The control groups of ten animals are fed 10% of the tota diet of non- transgenic foodstuff or crop material. A negative control group of ten animals are fed a diet that does not contain the crop material. The diets are balanced as closely as possible for total protein, carbohydrate, lipid and fiber. Animals are fed for sixty days. Water is provided ad libitum. Sera from the treated animals are used to assess allergenicity.

Preparation of antipen/allerpen: Collect the test materials in as pure form as possible, grind into a fine powder form and defat with ether (or hexane). Extract the defa‘tted material with buffered sahne, 10% wt./vol., however, this proportion can be varied depending upon the type of test compound. Mix the suspension at 20 to 25*C for 2 to 4 . hours to extract the proteins from the insoluble matter. Filter through Whatman No. 1 filter paper, then through 0.45 micron or smaller pore size sterile filters. Transfer to sterile vials and lyophilize or freeze the aliquotes at -8O’C.

Experimental Protocol The following in-vivo immunological assays could be used for the detection of reaginic antibodies in the test sera.

In-vivo assav: Passive Cutaneous Anaohvlaxis (PCAj Aonlication and limitations of test: PCA is an in;vivo method usually employed to assay the specific reaginic antibody present in serum. It is a useful,immunological tool to detect as little as 0.1 pg antibody protein. In this test the anaph’ylactic reaction is visualized as a local skin reaction.

Sex of animals: Male and/or female healthy young adult animals can be used. If females are used they should be nulliparous and non-pregnant.

Housing and feeding conditions: Where the lighting is artificial, the sequence should be 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used

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with an unlimited supply of drinking water. If guinea animals receive an adequate amount of ascorbic acid.

pigs are used, it is essential that

Preparation of the animals: Animals are acclimated to the laboratory conditions for at least 5 days prior to the test. Before the test, animals are randomized and assigned to the treatment groups. Removal of hair is done by close clipping, shaving or by chemical depilation. Care should be taken to avoid abrading the skin.

Principle: PCA could be produced-with the sera of the allergic host by challenging intradermaily sensitized sites with intravenously injected antigen/alIergen plus dye. Well-

‘defined blue areas appear, indicating the sites of antigen-induced extravasation of fluid due to the interaction with tissue fixed reaginic antibody.

Description of test nrocedure: Ndive animals are shaved on the back and flanks, avoiding abrasion of skin. Unblemished skin sites are selected and cleaned with 70% alcohol. Injections are spaced approximately 2-4 cm apart. 0. lml (or O.OSml) of test serum is injected intradermally. Doses are usually in the ratio of 1:2:4 or 1:3:9. The doses are injected using tuberculin syringe(s). The syringe used for the intradermal injection must not leak even under heavy injection pressure. Plastic disposable sterilized tuberculin syringes are satisfactorjl in this respect. Glass tuberculin syringes must be checked for leaks both at the needle butt and past the piston. Needles with “short bevel” points, usually i/z inch x 26 gauge are used. 4 to 72 hours later, 1 .Oml of antigen/allergen ( 1 mg or 2mg), in sterile physiological saline, with 1% Evans blue dye is injected intravenously or intraventricularly. For the intravenous injection the piston of the syringe must be easy moving so that there is no doub.t that the needle is in the vein when the smallest pressure is applied. The needle must be very sharp. For intravenous administration in guinea pigs, the vein which runs on the dorsal surface of the hind foot between the metatarsals of the outer and middle toes or ear vein or intracardiac’route can be used. In rabbits, the vein running along the margin of the ear is the most useful site but other veins which are easily accessible can also be used.

Assay design: Since cottonseed proteins are known allergens, the non-transgenic control sensitized animals should produce a significant reaginic antibody response. This assay is designed to measure differences in response of animals exposed to transgenic vs. non- transgenic cottonseed proteins by injecting equal doses @!Lsera from each group of animals into every naive PCA test subject. That way, each anirnal serves as it’s own control. In addition the ndive animals will receive sera from non-sensitized animals and from animals sensitized with a distinct line of cottonseed. The challenge antigen is also critical in this assay. One group of ndive animals that have been injected with serum dilutions will be challenged with the transgenic cottonseed proteins while a second group,

’ injected with identical serum diIution samples will receive a challenge dose from the non- transgenic cottonseed. Reciprocal comparisons of the differences in reaginic responses will be used to determine if there true differences in the immune responses induced by the transgenic plant relative to the non-transgenic plant, A third group,


injected with an identical serum pattern will not be chahenged with cottonseed protein, but will be injected with Evans blue dye to ensure that the extravasation is due to reaginic responses, rather than general irritation, or improper injection.

Observation:. 30-60 min. later, animals are killed. The skin is opened and reflected so that the lesions can be evaluated. Measurements of diameter and the assessment of intensity are usually made. This can be postponed until all the animals have been killed but the delay is not advised and should not exceed 2 hours. The skin must not be allowed to dry. Intensity of bluing is often expressed arbitrarily as + to +++. Since the relationship between the area of response and the dose is roughly linear, the plot of mean diameter upon log dose will also be linear. A table showing both would usually be preferred. When the potency of sensitizing antibody is unknown, a wider range of doses may be useful. The highest dose should give lesions of about 15 to 20 mm. Diameter and the smallest about 5mm.

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Appendix 7.2 Springborn Final Report

A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS

FINAL REPORT

Studv Director

Kimberly L. Bonnette, M.S., LATG

Studv Comoieted on

November 30, 1998

Performinq Laboratory

Springborn Laboratories, Inc. (SLI) Ohio Research Center

640 North Elizabeth Street Spencerville, OH 45887

SLI Studv No.

3044.697

Monsanto Studv No.

SB-98-212

Submitted to

Monsanto Company 800 N. Lindbergh Blvd.

St. Louis, MO 63167

Page 1 of 139

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SLI Study No. 3044.697 (2)

STATEMENT OF w CONFIDENTIALITY CLAIM

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4. ’ DATE

No claim of confidentiality is made for any information contained in this study on the basis of its failing within the scope of FIFFW lO(d)(l){A,), (B), or (C).

.

COMPANY:

COMPANY AGENT: g,‘c

.Crit&,t- - B;ob&. Req. i’c: TITLE

SLI Study No. 3044.697 (3)

COMPLIANCE STATEMENT

Study l%rector/Author Springborn Laboratories, Inc.

pLl?r.&L /,/ Date /2 2 /?9f

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SLI Study No. 3044.697 (4)

QUALITY ASSURANCE STATEMENT

This study was inspected by the Quality Assurance Unit and reports were submitted to management and the Study Director in accordance with SLl’s Standard Operating Procedures as follows:

Phase Date

Gene Check Assay 09/23/98 Data Audit 1 l/24/98,1 l/25/98,1 l/27/98 Report Review 11/30/98

Report to Study Director and Management

11/30/98

This study was conducted in compliance with the Good Laboratory Practice Standards as described by the EPA (40 CFR Part 160) and the FDA (21 CFR Part 58.)

(f&d&h& Anita M. Bosau, RQAP-GLP Director of Compliance Assurance

SLI Study No. 3044.697 (5)

TABLE OF CONTENTS

STATEMENT OF )KJ CONFIDENTIALITY CLAIM . . . . . . . . . . . . . . . . . , . . 2

COMPLIANCE STATEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . .a. . . . . . . . 3

QUALITY ASSURANCE STATEMENT ............................ 4

‘TABLEOFCONTENTS ....................................... 5 D

LlST OF TABLES AND APPENDICES ............................ 6

SUMMARY.. .............................................. 7

INTRODUCTION ............................................ IO

MATERIALS AND METHODS ................................. 10

EXPERIMENTAL PROCEDURES ............................... 13

ANALYSIS OF DATA ....................................... 16

MAINTENANCE OF RAW DATA, RECORDS ....................... 16

RESULTS ................................................ 16

CONCLUSION ............................................ 19

REPORT REVIEW ......................................... 19

REFERENCE .......................................... .:.2 0

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SLI Study No. 3044.697 (6)

LIST OF TABLES AND APPENDICES

Tables

1. Summary of Survival and Clinical Observations (Main Study) . 21 2. Summ.ary of Body Weight Data (Main Study) ............. 23 3. Summary of Body Weight Gain Data (Main Stildy) ......... 25 4. Summary of Hematology (Main Study) .................. 26 5. Summary of Biochemistry (Main Study) .................. 29

Appendices

A. Protocol and Protocol Amendment . . . . . . . . . . . . . . . , . . . . . 32

B. Results of the Feed Gene Assay . . . . . . . . . . . . . . . . , . . . . . 66

C. Certificate of Analysis (Ascorbic Acid) .................. 68 .

D. Clinical Pathology Methodology ....................... 70

E. Individual Clinical Observations ....................... 76

F. individual Body Weight Data (Main Study) ............... 92

G. Individual Body Weight Gain Data (Main Study) ........... 97

H. Individual Hematology ............................ “102

I. Individual RBC Morphology Data ..................... 113

J. Individual Biochemistry , , . . , . . . . . . . . . . . . . . . . . . . . . . . 119

K. Individual Gross Necropsy Observations (Main Study) . . . . . 130

L. Added Stock Animals . . . . . . . . . . . . i . . . . . . . . . . , . . . . . . 132 - .

M. SLI Personnel Responsibilities . . . . . . . . . . . . . . . . . . . . . . 138

SLI Study No. 3044.697 (7)

SUMMARY

This study was performed to provide an assessment of the allergenic activity of transgenic and non-transgenic cottonseed meal in a guinea pig model. Animals were td be sensitized by feeding diets containing cottonseed meal for approximately sixty days, to induce cottonseed protein specific IgG,,, the dominant reaginic antibody of guinea pigs. Antibody titers were to be measured by passive cutaneous anaphylaxis (PCA) following transfer of serum from sensitized animals to naive recipients. However, based on the results of the main study, in particular, the results pertaining to adverse ciinictil observationsireduced body weight/weight gain (possibly compromising PCA results) in conjunction with the results of the test feed analysis indicating that Group 1 did not receive test article feed through day 21, the PCA was not conducted. The main study design consisted of an untreated control group and three treatment groups with ten females in each group as follows:

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N6## 6312322 (Mechl Vans) 5713C-1 (lo%)* [5713C-I]‘* ,

N8## 6312323 (Mechl non-trans) 5713C-K (10%) I571 3C-K]”

NB## 6312331 (Mech3 non-trans) 5713C-M (10%) {5713C-M]”

Control Diet 5713C-H f5713C-Hl”

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*Apparently, from day 0 - day 21, the box utilized to feed this group was mislabeled by the manufacturer and was apparently control diet (5713-H) based on analytical results of the diet. A second box utilized beginning on study day 28 was the correct diet.

“Abbreviation of the test article name.

Animais were fed the appropriate pm-made diet as received from the Sponsor ad Iibitum from days O-21. Animals were returned to commercial guinea pig chow on day 21 through day 28 to allow for recovery from adverse clinical signs obsen/ed when feeding the test diets. On day 21, the animals were anesthetized with isoflurane and blood samples were obtained,ocularly (via orbital sinus) to conduct clinical pathology/clinical chemistry. In addition, blood samples were collected from five stock animals in order to compare the test diet animals with completely untreated animals. Animals were returned to their respective test diets on day 28. On day 30, water was provided ad libitum in water bottles (rather than the automated watering system) and supplemented with ascorbic acid (Vitamin C, 200 mg/t). Detailed clinical observations were conducted weekly and animals were observed daily for overt clinical signs of toxicity. individual body weights were obtained for the animals on study days 0, 7, 14, 17, 21, 24, 28, 35, 38, 42

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SLI Study No. 3044.697 (8)

and 49. Individual food consumption was measured three times a week ,during the study. On day 49, the study was terminated for both humane reasons concerning the palatability of the feed and the invalidation of Group 1 through day 21 due to the incorrect labeled feed. Gross necropsy examinations were conducted on animals that died or were euthanized for cause. No gross necropsy examinations were conducted for surviving animals.

One animal in Group 2 (Animal No. 4612) was fo&d-‘dead on day 27. The animals’s death was associated with poor feed ,intake/inadequate nutrition in conjunction with body weight loss and severe adverse clinical observations. This death may have been a direct result of Vitamin C deficiency as a consequence of an inadequate intake of Vitamin C sufficient diet, although this could not be definitively determined. A second animal in Group 3 (Animal No. 4648) was removed from study on day 28 due to severe adverse signs probably caused by the above. The onset of the majority of the adverse clinical observations began on day I.6 for Groups 2 and 3. These observation included: feces small in size, soft stool, rough coat, fecal stain, distended abdomen, dehydration, decreased food consumption and extremities biue in color (Vitamin C deficiency-like clinical observations possibly,due to reduced feed ,intake). After returning to commercial guinea pig chow on day 21, the majority of the clinical observations subsided. Upon study diet restart (day 28) along with the water bottles’ containing added Vitamin C (day 30), adverse clinical observations began to reappear by day 30 in all test groups (Group ?, 2 and 3). Due to the severity of clinical o,bservations causing an animal welfare concern (possible morta,lity) and due to an apparent lack of feed intake (probable palatability issue and/or inadequate diet), the study was terminated on day 49.

A statistically significant decrease in mean body weight was noted in Groups 2 and 3 by day 7 and continued to be decreased at days 14, 17; 21, 24 and 28. After returning to normal commercial guinea pig chow, body weight gains were similar from days 21 - 28. However, after resuming the test article diets, the body weight for Group 2 was significantly depressed by day 38 and both Groups 2 and 3 were significantly depressed by days 42 and 49. Although not significantly different, Group ‘l also had a notable decrease in body weight during this time period (day 35-49).

Examination of the hematology and biochemistry data revealed significantly decreased alkaline phosphatase, chloride, phosphorus, total protein and albumin in Groups 2 and 3 and significantly decreased globulin, *urea nitrogen, sodium and calcium in Group 3, only. Mean corpuscular volume and mean corpuscular hemoglobin were significantly decreased in the animals of both Groups 2 and 3. Hemoglobin and hemacrii were significantly decreased for Group 3, only. Alanine

SLI Study No. 3044.697 (9)

amino transferase was significantly increased in Group 2. There was a significant increase in segmented neutrophils, lymphocytes and leukocytes for Group 3. W,hen compared to the results of the stock animals, there were no notable differences: and, the results were only slightly outside published values for guinea Pigs [V.

Based on the results of this study, the most reasonable conclusion from body weight data, observations regarding feeding and clinical symptoms is that the 10% cottonseed diets provided by the Sponsor were unpalatable, or there was an undetermined toxic response to those diets specifically. The nearly immediate weight loss in ail cottonseed meal fed groups upon restarting the study on day 28 indicated that it was highly unlikely that the animals could have survived a full sixty day feeding trial on the appropriate diets. Because of the questionable health status and immune function of the surviving guinea pigs in this study, the Sponsor ordered the study terminated. Therefore, the PCA was not conducted since any resuits would not have been reliable utilizing serum from compromised main study animals.

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> SLI Study No. 3044.697 (10)

I. INTRODUCTION

This study was performed to validate that the Passive Cutaneous Anaphylaxis Assay (PCA) could determine the titer of IgG,, antibody present in serum of the animals fed (approximately 60 days) with test’article. However, based on the results of the main study, in particular, the results pertaining to adverse clinical observations/reduced body weight/weight gain (possibly compromising PCA results) in conjunction with the results of the test feed analysis indicating that Group I did not receive test article feed through day 21, the PCA was not conducted. This study was performed at Springborn Laboratories, Inc., 553 North &oadway, Spencerville, Ohio. The protocol was signed by the Study Director on July 24, 1998 (GLP initiation date). The in-life phase of the study was initiated with test article administration on July 27, 1998 (day 0) and concluded with termination on September 14, 1998.

II. MATERIALS AND METHODS

A. Experimental Protocol

The study protocol and protocol amendment are presented in Appendix A.

B. Test Articles

The test articles were received from the Sponsor and identified as follows:

Sponsor’s ID Ass;Ffb”d Physical

Description

5713GI Lot No.: 9848-2

S98.019.3044 Brown pellets Jynge93, None provided

5713GK Lot No.: 9848-4

S98.021.3044 Brown pellets June 9, None 1998 provided

5713C-M Lot No.: 9848-6

S98.023.3044 BrowTi/pellets June 9, None 1998 provided

57136-H’ Lot No.: 9848-l

V98.003.3044 Brown pellets June 9, None 1998 provided

*Apparently, from day 0 - day 21, the box utilized to feed this group was mislabeled by the manufacturer and was apparently control diet (5713C-H) based on gene feed analytical results of the diet. A second box utilized beginning on study day 28 was the correct diet (see Appendix B for results of the gene assay).

SLI Study No. 3044.697 (II)’

The test articles were stored refrigerated or at room temperature. The Sponsor is responsible for any necessary evaluations related to chemical composition, strength, purity, stability and other data required by 40 CFR 160.105 and 21 CFR Part 58.105. After study completion, each box of diet was anaiyzed by the Sponsor at SLI using a gene feed assay (See Appendix W

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C. Retention/Analytical Samples -

An approximate 20 g analytical sample of each lot of test/control article feed was taken on days 7, 14, 21 28, 35, 42 and at study termination (day 49) and stored in a -70’ C freezer for possible Sponsor evaluation. An approximate IO g retention sample of each lot of test/control article feed and each subsample was collected. These samples were stored refrigerated and serve as the retention samples for all studies conducted with the lot of test/control article feed utilized.

D. Test Article Disposition

The remaining test/control articles were dispensed of by incineration following completion of all studies with the test/control articles.

E. Method of Test Article Preparation

Each appropriate dietary test article for Groups 1, 2, 3 and 4 was fed as a pre-made diet as received from the Sponsor to each animal ad libitum. A Certificate of Analysis for each test article will be maintained by the Sponsor. On day 21, 50 g samples of each box of diet (3 boxes/test article diet) were j sent to Ralston Analytical Laboratories, 824 Gratiot, St. Louis, MO 63102.

F. Animals and Animal Husbandry

1. Description, Identification and Housing

Young adult, Hartley-derived albino guinea pigs (born on 6/19/98) were received at SLI from Charles Rivers Laboratories, a Inc. Raleigh, North Carolina. Upon receipt, plastic ear tags displaying unique identification numbers were used to individually identify the animals. Cage cards displaying at least the study number, animal number and sex were affixed

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SLI Study No. 3044.697 (12)

to each cage. The animals were housed individually in suspended stainless steel cages. All housing and care were based on the standards recommended by the Guide for the Care and Use of Laboratory Animals PI.

2. Environment

The animal room temperature and relative humidity ranges were 67-73°F and 38-80%, respectively. Environmental control equipment was monitored and adjusted as necessary to minimize fluctuations in the animal room environment. Light timers were set to maintain a 12-hour light/l2-hour dark cycle and room ventilation was set to produce IO-15 air changes/hour. The room temperature and relative humidity were recorded a minimum of once daily.

3. Food

PMI Certified Guinea Pig Chow #5026 (Purina Mills, Inc.) or diet provided by the Sponsor was provided ad libitum to the animals throughout the study. The lot number of each batch of diet used during the study were recorded. The feed was analyzed and certified by the supplier for nutritional components and environmental contaminants, Dietary limitations for various environmental contaminants, including heavy metals, pesticides, polychlorinated biphenyls and total aflatoxin are set by the manufacturer. Within these limits, contaminants which may have been present were not expected to compromise the purpose of this study. Results of the dietary analyses (Certificates of Analysis) are provided by the manufacturer for each lot of diet. Certificates of Anatysis from Purina Mills, Inc. are maintained by SLI. For the feed supplied by the Sponsor, Certificates of Analysis are maintained by the Sponsor.

4. Water

Municipal tap water treated by reverse osmosis was available ad libitum throughout the study. The purified water was supplied by an automatic watering system (day O-29) or reverse osr@osis water in water bottles supplemented with 200 mg/L ascorbic acid (Vitamin C, day 30-49). A Certificate of Analysis for the ascorbic acid was provided by the manufacturer and is presented in Appendix C. Monitoring of the drinking water for contaminants is conducted annually by SLI and the records are available for inspection, Within generally accepted limits, contaminants which may have been present were not expected to compromise the purpose of this study. The water meets the standards specified under the EPA National Drinking Water Regulations (40 CFR Part 141).

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SLI Study No. 3044.697 (13)

5. Acclimation

Upon. receipt, the animals were removed randomly from the shipping cartons, examined by qualified personnel, identified with plastic ear tags and then acclimated to the laboratory conditions for a minimum of five days. -The animals were observed daily for overt physical or behavioral abnormalities, general health/moribundity and mortality.

6. Animal Selection

Animals were weighed and examined in detail for adverse clinical observations. Animals determined to be suitable as test subjects were assigned to groups randomly by body weight. The animal numbers and the respective body weight values were entered into the computer. Homogeneity of groups by weight was the criteria of acceptance of the randomization. Disposition of animals not selected for study was documented in the study records.

III. EXPERIMENTAL PROCEDURES

A. Study Group Design

The main study consisted of :

Main Study No. of Test Group No. Animals

Administration (Days O-21 and Days 28-49)

1 10 NB# 6312322 (Mechl trans) 5713C-I (IO%)*

2 10 NB# 6312323 (Mechl non-trans) 5713C-K (10%)

3 10 Nf3# 6312331 (Mech3 non-trans) 5713C-M (10%)

4 10 Control Diet 5713C-H”*

*Apparently, from day 0 - day 21, the box utilized to feed-this group was mislabeled by the manufacturer and was apparently control diet (5713C-H) based on analytical results of the

diet. A second box utilized beginning study day 28 was the correct diet. “In addition, stock animals were assigned to two of the diets (Group 5: four animals to

5713C-I and Group 6: four animals to 5713C-H) from days 35-44 to determine if the results would repeat utilizing new animals.

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SLI Study No. 3044.697 ‘(14)

B. Dosing Procedures (Main Study)

1. Dietary Dosing

Each group of Dietary Test animals was fed the appropriate pre-made diet as received from the Sponsor ad libitum from day 0 through day 21. Due to severe adverse clinical observations/reduction in body weight/weight gain, the animals were removed from test/control diets on day 21 and resumed their appropriate test diet on days 28 - 49. During days 21-28 animals were fed PMI Certified Guinea Pig Chow #5026 in an attempt to return the animals to good health and complete the study. On day 35, *eight animals were chosen from stock to determine if new animals would respond the same way as the main study animals. Therefore, ,four animals were designated as Group 5 and fed diet 5713C-1 (Group I feed) and four animals were fed diet 5713C-H (IO%, Group 4 feed) until day 44 (day 9).

2. Clinical Observations

The animals were checked for general health/mortality and moribundity twice daily during the study. in addition, cage-side observations were performed once daily and detailed clinical observations were performed weekly.

3. Body Weights

Individual body weights were obtained for the animals on study days 0, 7, 14, 27, 21, 24, 28, 35, 3.8, 42 and 49. Note: Additional body weights were collected in order to evaluate animal health.

4. Food Consumption

Individual food consumption was measured 3 times a week. Food was also added as necessary to maintain ad libitum feed. Food consumption values were used as a means to track test article usage and are not reflective of the actual amount of diet consumptjon. Therefore, this data was invalidated as food consumption data asd will not be reported.

5. Collection of Blood SampleslCiinical Pathology

On day 21, animals were anesthetized with isoflurane and approximate 2 mL blood samples were obtained ocularly (via orbital sinus). Hematology and biochemistry parameters were evaluated (see Appendix D for methodology):

SLI Study No. 3044.697 (15)

a. Hematology Parameters

erythrocyte count (RBC) hematocrit (Hct) hemoglobin concentration (Hgb) mean‘corpuscular hemoglobin (MCH) mean corpuscular hemoglobin concentration (MCHC) mean coipuscular volume (MCV) platelet count reticulocyte count total and differentiat leukocyte counts

b. Biochemistry Parameters

aianine aminotransferase (ALT) albumin albumin/globulin ratio (calculated) alkaline phosphatase aspartate aminotransferase (AST) blood creatinine blood urea nitrogen (BUN) calcium cholesterol electrolytes (sodium, potassium, chloride) globulin (calculated) glucose phosphorus total biliru bin total serum protein

6. Euthanasia

Animals dying on study or euthanized moribund (carbon dioxide inhalation) during the study were necropsied. Body cavities (cranial, thoracic, abdominal and pelvic) were opened and examined. qn day 30, Animal No. 4648 was euthanized for cause. The proximal &.I distal ends of the femur and tibia were collected, processed/decalcified and evaluated by Dr. J. Dale Thurman, D.V.M., M.S., DACVP. No other tissues were retained. Following clinical observations and food consumption determination on study day 49, all surviving animals were euthanized by carbon dioxide inhalation or sodium pentobarbital and discarded.

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SLi Study No. 3044.697 (16)

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C. Protocof Deviations

Group 9 was fed the incorrect diet (control diet 5713C-H rather than 5713C-I) during the day O-21 interval due to a labeling error by the manufacturer. This error resulted in a compromised Group I. There was no expiration date for feed supplied by Sponsor. The animal room relative humidity range (38-80%) exceeded the preferred range (30-70%) during this study. The method of euthanasia for surviving animals was not documented. IACUC approval of this study was not obtained prior to study initiation. On day 0, the sample of each test/control feed article was inadvertently not collected. These occurrences were considered to have had no adverse effect on the outcome of this study.

IV. ANALYSIS OF DATA

Statistical analyses were performed using a MicroVax 3fOO computer. The level of significance was a minimum of 5% and tests were two-tailed. The control group data was compared to the treated data using all groups or by a group by group comparison depending on the test. Data including body weights, weight gain and clinical pathology were analyzed by one way analysis of variance. If significance was detected, a group by group comparison proceeded with Tukey- Kramer.

V. MAINTENANCE OF RAW DATA, RECORDS AND SPECIMENS

Ali original paper data, photographs, the final report, magnetically encoded records and any specimens were transferred to the SLI archives for a period of 7 years. The Sponsor will be contacted prior to final disposition of these items.

SLI Study No. 3044.697 (17)

VI. RESULTS

A. Survival and Clinical Observations

Table 1 (Summary Data) Appendix E (Individual Data)

One animal in Group 2 (Animal No. 4611) was found dead on day 27. The animal’s death was associated with poor feed intake/inadequate nutrition in conjunction with body weight loss and severe adverse clinical observations. This death may have been a direct result of Vitamin C deficiency as a consequence of an inadequate intake of Vitamin C sufficient diet, although this could not be definitively determined. A second animal in Group 3 (Animal No. 4648) was removed from study on day 28 due to severe adverse signs probably caused by the above. The onset of the majority of the adverse clinical observations began on day 16 for Groups 2 and 3. These observation included: feces small in size, soft stool, rough coat, fecal stain, distended abdomen, dehydration, decreased food consumption and extremities blue in color (Vitamin C deficiency-like clinical observations possibly due to reduced feed intake). After returning to commercial guinea pig chow on day 21, the majority of the clinical observations subsided. Upon study diet restart (day 28) along with the water bottles containing added Vitamin C (day 30), adverse clinical observations began to reappear by day 30 in all test groups (Group 1, 2 and 3). Due to the severity of clinicat observations causing an animal welfare concern (possible mortality) and due to an apparent lack of feed intake (probable palatability issue and/or inadequate diet), the study was terminated on day 49.

8. Body Weights and Weight Gain

Tables 2 and 3 (Summary Data) Appendices F and G (Individual Data)

A statistically significant decrease in mean body tightwas noted in Groups 2 and 3 by day 7 and continued to be decreased at days 14, 17, 21, 24 and 28. After returning to normal commercial guinea pig chow, body weight gains were similar from days 21 - 28. However, after resuming the test article diets, the body weight for Group 2 was significantly depressed by day 38 and both .Groups 2 and 3 were significantly depressed by days 42 and 49. Although not significantly different, Group 1 also had a notable decrease in body weight during this time period (days 35-49).

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SLI Study No. 3044.697 (18)

C. Collection of Blood Samples/Clinical Pathology

Tables 4 and 5 {Summary Data) Appendices H, t and J (individual Data)

. . .

Examination of the hematology and biochemistry data revealed significantly decreased alkaline phosphatase, chloride, phosphorus, total protein and albumin in Groups.2 and 3 and significantly decreased globulin, urea nitrogen, sodium and calcium in Group 3, only. Mean corpuscular volume and mean corpuscular hemoglobin were significantly decreased in the animals of both Groups 2 and 3. Hemoglobin and hemacrit were significantly decreased for Group 3, only. Alanine amino transferase was significantly increased in Group 2. There was a significant increase in segmented neutrophils, lymphocytes and leukocytes for Group 3. When compared to the results of the stock animals, there were no notable differences; and, the results were only slightly outside published values for guinea pigs [I].

D. Gross Necropsy Observations

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Appendix K (Individual Data)

Gross necropsy examinations of the animal in Group 2 (Animal’ No. 4611) which died on day 27 included body fat depletion, distended stomach and dark red apical lobe of the lung. The animal in Group 3 (Animal No. 4648), removed from study on day 28 due to severe adverse clinical observations/body weight loss and euthanized for cause on day 30, revealed body fat depletion, distended stomach and small intestines, mottled lungs, fluid in the abdominal cavity and foam in the trachea. A necropsy examination was not required for surviving animals. Also the proximal and distal ends of the femur and tibia appeared within normal limits to the board-certified pathologist.

E. Added Stock Animals

Appendix L (Individual Data)

Within two days post feeding, all four of the stock animals in Group 5 fed 5713C-I (same diet as Group 1) developed few feces and/or soft stools and/or ,feces small in size. In addition body weight loss/reduced body weight gain was observed in all four of the stock animals. No body weight loss was observed in Group 6 fed control diet (5713C-H same diet as Group 4). These resuits were similar to the effects observed when administering the correct test

SLI Study No. 3044.697 WJ)

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diet to Group 1 on day 28. The observations associated with feeding this additional group of animals the cottonseed meal diet confirm the findings in the main study, that the cottonseed diets appear to be the direct cause of the health problems that occurred in feeding Groups I, 2 and 3.

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VII. CONCLUSION

Based on the results of this study, the most reasonable conclusion from body weight data, observations regarding feeding and clinical symptoms is that the 10% cottonseed diets provided by the Sponsor were unpalatable, or there was an undetermined toxic response to those diets specifically. The nearly immediate weight loss in all cottonseed meal fed groups upon restarting the study on day 28 indicated that it was highly unlikely that the animals could have survived a full sixty day feeding trial on the appropriate diets. Because of the questionable health status and immune function of the surviving guinea pigs in this study, the Sponsor ordered the study terminated. Therefore, the PCA was not conducted since any results would not have been reliable utilizing serum from compromised main study animals.

Study Director

VIII. REPORT REVIEW

Deborah A. Douds, M.S. Assistant Manager of Acute Toxicology

Date

\ 4L jdL-q4c . Siglin, Ph.D., j$ABT

Director of Toxicology

Date

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SLI Study No. 3044.697 (20)

IX. REFERENCES

1. Terrel, L. A., D.V.M., MS. and D. J. Clemons, D.V.M., M.S., The Laboratory Guinea Pig, ed. M. A. Suckow, .D.V.M., CRC Press LLC, 1998, pp. 21, 22 arid 61-63.

2. Guide for the Care and Use of Laboratory Animals, DHHS Publication No. (NIH) 96-03, 1996,

SLI STUDY NO.: 3044.697 TABLE 1

A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS PAGE 1 CLIENT: FlONsANTGcoKPANY SUBliARY OF SURVIVAL AND CLINICAL OBSEWATIONS (OCCURREXE/ANIHALS AFFECTED)

. ..e-__ F E H A L E -e-m-

--------------------_____^______________----------------------------------------------------------------------------------------- TABLE RANGE: DAY 0 'I-0 DAY 49

GROUP: 1 2 3 4 LEVEL: 5713C-I 5713C-K 5713C-H !5713C-H

--------------------------------------------------------------------------------------------------------------------------------- NORMAL -NO REBAREADLE CLINICAL OBSERVATIONS 72/10 58/10 66/10 76/10

DEAD -FOUND DEAD o/ 0 I/ 1 o/ 0 o/ 0 -REMOVED FROMSTUDY, BUTNOT EUTHANIZEDAT'IZIS o/ 0 o/ 0 I/ 1 o/ 0

- TIHE -SCHEDULED EUIHANASIA lO/lO 91 9 9/ 9 1000

ACTIVITY -SALIVATION o/ 0 o/ 0 l/ 1 o/ 0

EXCRETA -FEW FECES 12/ 7 33/ 9 30/ 7 lo/ 7 -FECES SHALL IN SIZE 9/ 7 93/10 531 9 14/ 8 -SOFT STOOLS s/ 5 a/ 5 9/ 4 13/ a

BODY -URINE STAIN 3/ 3 13/ 3 4/ 3 4/ 3 -ROUGH COAT o/ 0 44/ 9 30/ 5 o/ 0 -FECAL STAIN 151 a ‘7/ 4 2/ 1 12/ 4 -EXTREMITIES APPEAR BLUE IN COLOR o/ 0 12/ 3 5/ i 01 0 -DISTENDED ABDOHEN o/ 0 24/ 4 7/ 2 o/ 0 -DEHYDRATION o/ 0 2/ 1 2/ 1 01 0 -HAIRLOSS o/ 0 l/ 1 o/ 0 o/ 0

EYES -DARK HATERIALAROUND EYE(S) o/ 0 o/ 0 l/ 1 o/ 0 -------------------------------------------------------------------------------------------------------------------------- ---m-w- NOTE: DATAREFLECTTKETOTALOCClJRWKEOF EACHCLINICALFINDINGOVER ‘IHEMJHBEROF ANIMLS EXHIBITI& THE FINDING.

TABLE 1 SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GiJINEh PIGS PAGE 2 CLIENT: HWSANTU COKPANY S-Y OF SURVIVAL AND CLINICAL OBSERVATIONS (OCCURRF.NCE/AN~ AFFECTED)

-m-e_ F E H A L E ..--.s-

-------------L-------------------------------------------------------------------------------------------------~-----------------

TABLE RANGE: DAY 0 To DAY 49 GROUP: 1 2 3 4 LEVEL: 5713C-I 5713C-R 5713C-N 5713C-H

---L----------------___________I_______I---------------~---------------------------------------------------------- __-c-_--.mI--^-- QUALT FOOD/UATER -DECREASED FOOD CONSUHPTION 4/ 3 41/ 9 15,' 6 4/ 4 -DECREASED WATER CONSUtfPTION 4/ 4 29/ 5 a/ 2 21 2 -----------L--------_____^______________--------~-------------------------------------------------------------------------------- NOTE : DATA REFLECT THE MTAL OCCURRENCE OF EACH CLINICAL FINDING OVEEl ‘JXE NUKBEX OF ANIMALS EXIIIBITING THE FINDING.

SLS STUDY NO. : 3044.697 CLIENT: tmNsm COHPANY

TABLE 2 A PASSIVE tXlTMEWS ANAFliYLAXIS ASSAY IN GUINEA PIGS

SUHHARY OF BODY WEIGHT DATA (GRAHS) PAGE 1

- ___a F E H A L E e-_-w

-------------------_________lfC_________-----------------------------------------------------------------------------------------

GROW: 4 2 3 LEVEL: 5 7 13C-H

5713c-: 5713C-K 5713C-H

------------------------------------------------------------------------------------------------------------*-------------------- DAY 0 HEAN 333 332 333 333

S.D. 10.3 : 10.8 11.1 10.4 N 10 . 10 10 10

DAY 7 HEW 366 379 342~ 339* S.D. 16.4 16.3 21.0 8.9

N 10 10 10 10

DAY 14 HEAN 408 408 346# 3568

S.D. 23.6 18.7 25.3 11.3 N 10 10 10 10 E

DAY -17 HEAN 421 419 3468 345% S.D. 27.9 27.3 22.2 ’ 11.2

N 10 10 10 10

DAY 21 HEAN 444 443 355# 35911 ' S.D. 29.6 31.2 ‘22.2 13.6

N 10 10 10 10

DAY 24 HEAN 461 452 3735 381# S.D. 31.4 27.4 34.3 25.8

N 10 10 10 LO

DAY 28 HEIN 484 473 405# 4028 S-D. 36.0 29.1 31.6 32.8

N 10 ! 10 9 LO -------------------I____I_c1____________----------------------------------------------------------------------------------------- SIGHIFICANTLY DIFFEREM? FROM CONTROL: ** = P<O.Ol # = P<O~OOl

J I I I I

SLS STUDY NO.: 3044.697 CLIE3dT: ffmsm COHPANY

TABLE 2 A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS

S-Y OF BODY UEIGKC DATA (GRAHS) PAGE 2

w-e..- F E H A L E --e-e

-------c---------------“----------------------------------------------------------------- ^-^--I-----c--------_______II_____ _-------

GROUP: 4 1 2 3 LEVEL: 5713C-H 5713thI 5713C-K 5p3c-n

--------------------_______^____________----------------------------------------------------------------------------------------- DAY 35 HEAN 501 453 403x 41M

S.D. 47.5 45.2 49.7 25.6 N 10 10 9 9

DAY 38 HEAN 525 479 41611 43711 S.D. 43.4 40.0 60.5 33.2

N 10 10 9 9

DAY 42 MEAN 545 508 4348 4565 S.D. 41.9 31.7 58.4 28.4

N 10 10 9 9

DAY 49 HFAN 551 529 4435 466~ S.D. 51.7 48.1 54.7 32.8

N 10 10 9 9

DAY 56 nut4 S.D.

N

DAY 60 NEXN S.D.

N --------------------__CI_C_______f______---------------------------------------------------------------------------------- _------ SIC;NIFICANTLY DIFFERENT FROH CCMTROL: e = P<O.Ol # = P<O.OOl

-. .-.

SLS STUDY NO.: 3044.697 CLIENC: HONSANTO COHPANY

TABLE 3 A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS

SUtCHARY OF BODY WEIGHT GAIN DATA (GRAHS) PAGE 1

-.-c-e F E H A L E ,a.---- -------------------------------------------------------------------------------------------------------------~-------------------

GROUP: 4 .5713c-:

2 3 LEVEL: 5713C-H 5713C-K 5713c-w

-------------------I-----------------------------------------------~------------------------------------------------------------- DAY OTO 7 HEAN 33 47* 9P 611

S.D. 8.8 7.5 16.6 6.2 N 10 10 10 10

DAY 7m 14 HEAN 42 29* 411 1711 S.D. 9.5 9.6 11.1 10.7

N 10 10 10 10

DAY 14 To 21 HEAN 35 35 98 J 311 S.D. 13.4 14.5 11.9 14.9

N 10 10 10 10

DAY 21 M 28 nut4 40 30 50 43 S.D. 19.2 7.0 14.6 24.0

N 10 10 9 10

DAY 28 M 35 HEhN 17 -19 -3 -1 S.D. 29.5 33.3 30.1 20.0

N 10 10 9 9

DAY 35 TO 42 WEAN 44 55 31 45 S.D. 28.9 26.5 24.8 28.5

N 10 10 9 9

DAY 42M 49 HJiXN 6 21 9 10 S.D. 16.5 21.2 16.9 14.8

N 10 10 9 9 --------------------__________________I_-----------------------------------------------------------------------------------------

SIGNIFICAWLY DIFFENNI' FROM tX,KCROL: * = P<O.O5 11 = P<O.OOl

i I I

SLS STUDY No.: 3044.697 CLIFNr: HoNSAElTo COHPANY

TABLE4 A PASSIVE CUTANEOUS ANdPHYMXIS ASSAY i?? GUINEA PIGS

SUHHARY OF HMAMLDGY DATA PAGE 1

___-_ F E H A L E ---em

-c------------------------------------------------------------------------------------ __c-------*-.--^--.. ______-_-^----------^____ GROUP: 4 2 3 LEVEL: 5713C-H 5713C-: 5713C-K :5713ca

----------------------------------------------------------------------------- ___________________________^________^___------------ ERYTKROCYTES

DAY 21

Hl?tlDGLDBIN DAY 21

HFiWNKRIT DAY 21

NEXN CORPUS VOL DAY 21

HCH DAY 21

HCHC DAY 21

10*6/CHH HEAN 5.05 4.80 5.03 4.77 S-D. 0.232 0.279 0.323 0.313 N 10 10 10 10

G/DL

%

FL

PC

G/DL

13.4 13-o** 0.69 0.95

10 10

HEAN 14.3 13.7 S.D. 0.68 0.84 N LO 10

39. o** 2.89

10

HEAN 43.1 41.5 S.D. 1.87 2.64 N 10 10

40.9 2.10

10

81.48 81.8** 2.11 1.30

10 10

HEAN 85.3 86.5 S.D. 2.41 1.96 N 10 10

26.7# 27.2~ 0.65 0.55

10 10

HEAN 28.2 28.5 S.D. 0.89 -0.38 N 10 10

33.0 32.8 0.44 q-47

10 : 10

33.3 0.47

10

HEAN S.D. N

33.1 0.35

10 --------_-__--__-_______________________--------------------------------------------------------------------------------- _^------

SIGNIFICANTLY DIFFERENT FROH CXMXOL: * = P<O.Ol # = P<O.OOl NOTE : THE HEX6 AND STANDARJI DEVIATIONS WERE CALCIJLATEB USING NOblEtOUNDE3l VALUES.

SLS STUDY NO.: 3044.697 CLIENT: HONSANMCOKE'M

TABLE 4 A PASSIVE CUTANEOUS ANAPHYihXIS ASSAY IN GUINEA PIGS

SUHHARY OF HEXA'IBblGY DATA PAGE 2

-c--v F E H A L E -e--w

-------------1-_-_--___l______l_________------------------------------------------------ __,,,__-_,,,,--,,,,---~----------- -..----.“.

GROUP: 4 1 2 3 LEVEL: 5713C-H 5713C-I 5713C-K ,5713C-H

--------------------_________________c__------------------------------------------------ -_-----c---_---__-I-_______I____^_______-

PLATELETS 10*3/CHH DAY 21 HEAN

S-D. N

556 119.7

10

454 493 103.9 119.5

10 10

470 90.6

10

SEGD NFiUTROPHILS X WBC DAY 21

LYKPHOCYTES x WBC DAY 21

HONOCYTES % WBC DAY 21

BASOPHILS % WBC DAY 21

EOSINOPHILS DAY 21

% WBC

HEAN 30 S.D. 10.4 N 10

nut4 S-D. N

HEAN S-D. N

nEAN S.D. N

HEAN S-1). N

68 61 44* 36f 9.8 18.8 18.8 16.4

LO 10 10 10

1 0.7

10

0 0.3

10

2 1.3 10

36 55*x 62# 17.8 18.8 17.1

10 10 10

1 2 0.9 1.0 10 10

2* 1.2

10

0.: 0 0

0.0 0.0 10 10 10

2 0 1 2.0 0.3 0.7

10 10 10 ----------_------_-__c__________c_______-----------------------------------------------------------------------------------------

SIGHIFICANTLY DIFFERENT FROH CONTROL: * = P<O.OT ** = P<O.Ol I/ = P<O.OOl NOTE : THE MEANS AND STANDtiZl DEVIATIONS WERE CALCULUED USING NONROUNDED VALIJES.

I I 1 I

SLS STUDY NO.: 3044.697 CLIENT: HaNSrn COHPANY

TABLE 4 A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUm PIGS

SUHHARY OF HEHA'lXXOGY DATA PAGE 3

,.

___-_ F E H A L E -e-w-

--------------------__________^_^___I___-----------------------------------------------------------------------------------------

GROUP: 4 1 2 3 LEVEL: 5713C-H 5713C-I 5713C-K 5713C-H

__^___ --------------------__1______1_1__^_____----------------------------------------------------------------------------------- NON-SEGD NEXTRO. X WBC

DAY 21 HEAN - 0 0 0 0 S-D. 0.0 0.0 0.0 0.0

N 10 10 10 10 .

NUCLEATED RBC'S % WBC DAY 21 HEAN 0 0 0 0

S-D. 0.0 0.0 0.0 0.0 N 10 10 10 10

RETICULOCYTES % lu3c DAY 21 MEAN 1.8 2.1 1.6 1.2

S-D. 0.70 0.97 0.74 0.58 N 10 10 10 10

LEUKOCYTES 10*3/cMM DAY 21 HEAN 6.44 7.09 12.00* 14.22**

S-D. 0.989 2.671 5.043 6.187 N 10 10 10 10

-------------------1___________________I------ _-_______c__c__-____--------------------------------------------------------------- SIGNIFICANTLY DIFFERENT FROH CONTROL: * = P(O.05 j;j: = P<O.Ol NOTE: THE HEANS AND STANDARD DEVIATIONS WERE CALCULATED USING NONROUNDED VALUES. .

'S3RTVA U3ClNJlOuEKIM SNISn (LElWXlW3 3?EIA SNOLWIA3U tTlWQNV,TS UNV SmH 3KL :XT,ON TOO-Ox = ii '10'0>d = 4% S0'0>d = + :?t)kLlN# W&T tliN3EIddIff A7.7.WXdl~IS __________-___--__--_______^______l___ -_--__---_--I--^-_-------------------------------------------------------------------------

07 OT OT ZLO’O OTT -0 7LO'0 8E'0 09-a 6E'0

07 f 8EO'0 6E'0

N -a-s Nvm

1cmH 'I2 hva 3NINlLm

lz Ava ?OlT3JS31OH3

tz hV0 3aIuulm

TZ hVU HlKTIV3

TZ AVU N3mH4TlIN v3w

TZ hVU 3SVsLSOHd XTV

OT 7-8 +7E

OT E-n 8E

OT 6-L 05

01 7'11 59

N -a-s Nv3N

1a/3u

01 OT Z-Z T-E

i/807 *Ott

0t 2-z EZT

01 5-z 917

N -a-s Nvm

l/lOHH

01 ETE'O

“YZL l 07

O'i TO%-'0 9t 'TT

01 TSS'O 60'71

01 8TE'O 8E'T‘I

N -a's mw

lam

01 01 S-T 6-O 6T 12

OT 0-l 9-z 0'2

#ZT 67

N ‘U’S Nvm

Tam

OT OT P'8Z f’OZ

tttzt #&ZT

OT P'EE 782

07 &'TE 08T

N -a-s Nv3H

1m.T ,._^_-_,..---..--I..-.. _______I______C--_--______L___________^_------------------------------------------------------------------------

KXTLS ,’ XIE’ILS I-3EILS H-3EIL5 wmrT E 2 T )I : dnol33

------~-----^------------------------------ -____cI--------_------------*------ --------------------^___________________"---------- ----- 3 1 v H 3 d. ----- .

KLVC.7 AUSIWH3oIB d0 hliIVHMlS ANvdHo3 lI@NvSNoH xN3173 7 33Vd S3Id vmm3 NI AYSsv SIx\pz;IHl7vNv sno3wm 3AISSVd 76 L69 l )l’iOE :-ON ACtALS SlS

s 3lmTi

TABLE 5 SLS STIJDII NO.: 3044.697 A PASSIVE CIXJTANEOIJS ABAE'BYLAXIS ASSAY IN GUINEA PIGS CLIEW: HrnSAEIM COHPANY SUHHARY OF BIOGBEUISTRY DATA

PAGE 2

we--- f E H A L E --__..

--------------------------------------------------------------------------~-----------~------------------------------------------

GROUP: 4 I 2 3 LEVEL: 5713C-H 5713c-I 5713C-K 5713C-H

--------------------------------------------------------------------------------------------*--------------,---------------------- ASP. AHINOTRANS. IU/L

DAY 21

POTASSIUH DAY 21

SODIIJB DAY 21

PHOSPHORUS DAY 21

TOTAL BILIRUBIN DAY 21

TOTAL PROTEIN DAY 21

HEAN 54 72 S.D. 19.2 46.7 N 10 10

04 89 23.4 28.1

10 10

HHOL/L HEAN S.D. N

5.11 5.16 4.96 4.71 0.643 0.423 0.373 0.466

10 10 10 10

HBOL/L HEAN S.D. N

141 140 140 138~ 1.6 1.8 2.8 1.8 10 10 10 10

HG/DL HEAN S.D. N

5.78 7.1 0.80

10

6.6 > 0.59

10

5.58 0.55

10 0.64

10

HG/DL HEAN S.D. N

C/DL * HEAN S.D. N

0.47 0.50 0.54 0.56 0.105 0.080 0.135 0.063

10 10 10 10

4.90 4.73 4.52* 4.04c 0.264 0.212 0.385 0.263

10 10 10 10 --e-e --------------------______________I_____------------------------------------------------------------------------------------

S1GIiIFICANM.Y DIFFERENT FROH OONTROL: * = P<O.O5 ** = P<O.Ol # = P<O.OOl NOTE: THE HEANS AND STANDARD DEVIATIONS WRE QJ.,GULATED USI& NONR(XJNDEB VALUES.

SLS STUDY NO.: 3044.697 CLIENT: HONSANTO COMPANY

TABLE 5 A PASSIVE CLKANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS

SUNHARY OF BIOCHEHISTRY DATA PAGE 3

_a-_- F E H A L E v-ma..

-------------------_------------------------------------------------------------- __^____-__------_-__----------------------------

GROUP: 4 1 2 3 LEVEL: 5713C-H 5713C-I 5713GK 5713C-H

-------c--------___________cI___________---------------------------------------- ------------------------------c--- ----v--- ------- A/G RATIO

DAY 21 1.04 1.08 0.071

10

HEAN 1.05 1.08 S.D. 0.090 0.077 N 10 10

0.104 10

GLUCOSE HG/DL DAY 21 MEAN 149 149 146 147

S.D. 13.2 24.7 9.9 14.7 N 10 10 10 10

GMBULIN DAY 21

G/DL 2.22 1.95/k

0.288 0.150 10 10

HEAN 2.40 S.D. 0.190 N 10

2.28 0.119

10

ALAN.hHItWlXANS IWL DAY 21 34

9.3 10

68** 57 38.6 16.2

10 10

HEAN 33 S.D. 5.1 N 10

ALBUHIN G/DL DAY 21 HEAN 2.50 2.45

S.D. 0.146 0.151 N 10 .lO

2.30* 2.09% 0.138 0.145

10 10 ---------------__-_------------------------------------------------------------------------ ___^___------------------------------- SIGNIFICXdTLY DIFFERENT FROH CXMTROL: * = P<O.OS * = P<O.Ol # = P<O.OOl NOTE: THE HEANS AND STANDARD DEVIATIONS WERE CALCULATED USING NONROUNDED VALUES.

SL\ Study No. 3044.697 (32)

APPENDIX A

Protocol and Amendment

in

(33)

-

A PASSIVE CUTANEOUS ANAPHYLAXIS ASS@!’ IN GUINEA PIGS

-

Springborn Study No. 3044.697 Monsanto Study No. SB-98-2-I 2 -

-

Springborn Laboratories, 11%. (SLI) Ohio Research Center

640 North Elizabeth Street Spencerville, Ohio 45887

Kimberly L. Bonnette, M.S., LATG Study Director

For

Monsanto Company 800 N. Lindbergh Blvd. St. Louis, :MO 63167

-

ak t ,

i a

i . 1

pe

b”

1 & I

SLI Study No. 3044,697

TABLE OF CONTENTS

PURPOSE :: ................................................... 1

RESPONSISILIVES .............................................. 1

PROPOSED STUDY SCHEDULE ................................... 2

TEST ARTICLE IDENTIF1CATION ................................... 3

TEST SYSTEM . . . . . . . . . . . . .

EXPERIMENTAL DESIGN AND

PROTOCOL AMENDMENT . . ,

,..........*.*..............,..+...., 5

PROCEDURES ........................ 9

.................................... 13

DATA REPORTING .............................................. 14

ANALYSIS OF DATA ............................................ 14

MAINTENANCE OF RAW DATA, RECORDS AND SPECIMENS .......... 15

REGULATORY COMPLIANCE .................................... 15

QUALITY ASSURANCE .......................................... 15

USDA ANIMAL WELFARE COMPLlANCE STATEMENT ................ 16

DECLARATION OF INTENT ...................................... 16

PROTOCOLAPPROVAL .......................................... 17

REFERENCES ................................................. 18

PROTOCOL APPENDIX

A. Figure1 ............................................ ..I 9

t ,

{ii)

'(35)

SL1 Study No. 3044.697 Page1 of19

1. PURPOSE -

The purpose of the study is to determine by Passive Cutaneous Anaphylaxis Assay the titer of IgG;, mtibody present in the serum of animals fed test article (approximately 60 days).

A.

E3.

C.

cl.

Il. RESPONSlSlLITlES I

Sponsor’s Representative -

Sponsor

Monsanto Company 800 N. tindbergh Blvd. St. Louis, MO 63167

Terry Kaempfe Phone: (314) 694-1318 Fax: (314) 694-4028 E-mail: terry. kaempfe@monsanto. corn

Study Monitor

Rick Goodman, Ph.D. Phone: (314) 737-5314 Fax: (314) 737-6289 E-mail: [email protected]

Testing Location

Springborn Laboratories, Inc. Ohio Research Center 553 North Broadway Spencerviile, OH 45887 Telephone: (419) 647-4196 FAX: (419)647-6560

-

(36)

Stl Study No. 3044.697 Page2of19

D.

A,

6.

C.

Personnel Responsibilities

1.

2.

3.

4.

5.

6.

7.

8.

9.

Kimberly i. Bonnette, M.S., LATG Study Director/Manager of Acute Toxicology E-mail: [email protected]

George A. Douds, M.S. Alternate Contact/Assistant Toxicologist

Deborah A. Douds, MS. Assistant Manager of Toxicology

Robert C. Springborn, Ph.D. Chairman, President and CEO

Malcolm Blair, Ph.D. Vice President and Managing Director

Jo.seph C. Siglin, Ph.D., DABT Director of Toxicology

Rusty E. Rush, M.S., LAT, DABT Associate Director of Toxicology

J. Dale Thurman, D.V.M.,, M.S., DACVP Director of Pathology

Anita M. Bosau, RQAP-GLP Director of Compliance Assurance

Ill. PROPOSED STUDY SCHEDULE

Initiation of In-Life Phase: July 1998

Completion of In-Life Phase: August 1998

Audited Report Date: 6 Weeks Following Sponsor’s Approval for Study Termination

(37)

SLI Study No. 3044.697

IV. TEST ARTICLE IDENTtFICATlONS

A. Test Articles

Page3ofl9

.,

1. Main Study/PCA Assay

“Phase

Dietary Dosing [Group 1)

Sponsor Identifications

NB# 6312322 (Mechl trans) 5713C-1’ (10%)

SLI Identification Nos.

S98.019.3044 .

Dietary Dosing (Group 2) NB# 6312323 (Mechl non-trans) 5713C-K (10%)

S98.021.3044

Dietary Dosing (Group 3) NB# 6312331 (Mech3 non-trans) 5713C-M (10%)

S98.023.3044

Dietary Dosing (Group 4)

PCA IC’ Dosing

PCA IC Dosing

Control Diet 5713CH

t.*

*t*

V98.003.3044 *we

f.,

PCA lC Dosing

PCA ID” Dosing

~-.~ --~ .*I

Histamine

*t*

598.007.3044

PCA IC Dosing Evans blue (crystals from Sigma) I S98.012.3044

PCA ID/IC Dosing Sterile Saline V98.006.3044

*tntracardial, ‘*lntradermal, ***To be added by protocol amendment.

2. Characteristics

The Sponsor is responsible for any necessary evaluations related to chemical composition, strength, purity, stability and other data required by 40 CFR 160.105 and 21 CFR Part 58.105. Any special storage conditions fcr the test/control articles will be supplied by the Sponsor, The Sponsor has requested that the bulk feeds be maintained refrigerated (-4°C) and smaller amounts (subsamples) removed weekly or as necessary (to be maintained refrigerated) for dispensation.

r 3. Handling Precautions

-

-

-

-

Safety data regarding the test/control articles should be provided by the Sponsor [Material Safety Data Sheet (MSDS) or equivalent, if avaitabte]. Technical personnel are required to read this information prior to handling

(38)

SLI Study No. 3044.697 Page4of19

c i

F

the test/control articles. Any question concerning this information should be referred to the Study Director.

.P

.c. k

h* J

D.

yt

b *

Additional safety and handling information may be provided by the Study Director and/or Sponsor. Minimum safety requirements include safety glasses, impervious gloves, and laboratory wear. An MSDS shall also be available for any chemical entities utilized in the conduct of this study.

Analytical/Retention Samples

An approximate 20 g analytical sample of each lot of test/control article feed (Groups l-4) will be taken on days 0, 7, 14, 21, 28, 35, 42, 49, 56 and at termination and stored in a -70°C freezer for possible Sponsor evaluation. An approximate 10 g retention sample of each test/control article feed will atso be collected from each bulk test/control article and from each subsample. The sample wiii serve as the retention sample for ait studies conducted with the lot of test article utilized.

Test/Control Article Disposition

All remaining testlcontrol articles will be returned to the Sponsor following completion of all studies with the test/control articles unless otherwise instructed by the Sponsor.

Method of Dosage Preparation for Injection Animals

1. Main Study

a. Dietary test/control article (day 0 through day 60 f 2 days)

Each appropriate dietary test/control article for Groups 1, 2, 3 and 4 will be fed in a premade diet as received from the Sponsor to each animal ad libitum. The Sponsor will provide analysis to be included in the final report. The method of preparation may be provided by the Sponsor, documented in the raw data and presented in the final report.

(39)


. . .2. PCA Assays

a. Histamine for PCA Assay

Page5of19

Histamine will be administered intradermally at a I:10 dilution in sterile saline or as directed by the Sponsor. The test article will be prepared and/or dispensed fresh on the day of dosing. The method of preparation will be documented in the raw data and presented in the final report.

-\

b. Sterile Saline

Sterile saline may be administered intradermally as received (if requested) and may be dispensed on the day of dosing. The method of preparation will be documented in the raw data and presented in the final report.

c. Evans blue

An Evans blue solution of 6% w/v in sterile saline will be prepared for use on each day of the PCA Assay. The method of preparation will be documented in the raw data and presented in the final report.

d. PCA Challenge Solutions

To be determined and added by protocol amendment.

V. TEST SYSTEM

A. Justification of the Test System

1. The guinea pig is the preferred species for immunological testing by various U.S. and International regulatory agencies.

2. The Hartfeyderived albino guinea pig has been shown to be sensitive to the hyperallergenic effects of a variety of drugs and chemicals. Therefore, this species and strain is a reasonable alternative to larger mammals for dermal sensitization testing of drugs and chemicals for human safety assessment.

(40)

SLI Study No, 3044.697 . . Page6of19

3. The’Hartley-derived albino guinea pig has been used extensively for dermal sensitization testing. Thus, data from this study may be compared and contrasted to other studies performed in Hartley-derived albino guinea pigs.

4. Historical information concerning Hartley-derived albino guinea pigs is availabie at SLI and in the published literature.

5. Healthy, outbred Hartley-derived albino guinea pigs may be obtained from reliable, USDA approved and regulated suppliers. .

6. The laboratory guinea pig may be safely handled and manipulated by trained technical personnel.

P

6. Justification of the Route of Exposure and Number of Animats

1. Feeding was selected as a potential route of human exposure of the antigen being evaluated.

2. Since Hartley-derived albino guinea pigs are PVR antibody free, the response should be specific for the antigen.

3. The number of animals used on this study will be the minimum number to ensure scientifically meaningful results and to allow for adequate practice for the future studies.to be conducted.

C. Description _

1. Species

Guinea Pig I”I

1 d

2. Strain

Hartley-derived albino, VAF

3. Source

Charles River Laboratories, Inc. or another USDA approved supplier


(41)

Page7of19

,-

D.

E.

4. Age &d Body Weight Range

Young adult, Main Study Animals will weigh approximately 250 g to 500 g (on day 0) and PCA animals approximately 250 to 500 g (on the day prior to dosing).

5. Number of Animals/Sex on Study

All female guinea pigs

40 Main study animals (IO per test group; 4 groups)

A maximum of 90 animals will be utilized for the PCA assay. The actual number will be documented in a protocol amendment.

Method of Identification

Plastic ear tags displaying unique identification numbers will be used to individually identify the animals. Cage cards displaying at least the study number, animal number, and sex will be affixed to each cage.

Animal Husbandry

1; Housing

The animals will be housed individually in suspended stainless steel cages. All housing and care will be based on the standards recommended by the Guide for the Care and Use of Laboratory Animals [+I]+

2. Environment

The environmental conditions for the animal room will be set to maintain room temperature and relative humidity ranges of 70 k 7°F and 50 C 20%, respectively. Environmental control equipment will be monitored and adjusted as necessary to minimize fluctuations in the animal room environment. Light timers will be set to maintain a 12-hour light/l2-hour dark cycle and the room ventilation will be set to produce 1 O- 15 air changes/hour. The room temperature and relative humidity will be recorded a minimum of once daily,

.I

.)L


3. Food

PM1 Cqtified Guinea Pig Chow #5026 (Purina Mills, Inc.) or diet provided by the Sponsor will be provided ad libitum to the animals throughout the study. The tot number and expiration date of each batch of diet used during the study will be recorded. The feed is analyzed and certified by the supplier for nutritional components and environmental contaminants. Dietary limitations for various environmental contaminants, including heavy metals, pesticides, polychlorinated biphenyls and total aflatoxin are set by the manufacturer. Within these limits, there are no contaminants reasonably expected in the diet which would interfere with the conduct of the study. Results of the dietary analyses (Certificates of Analysis) are provided by the manufacturer for each tot of diet. These wili he maintained in the laboratory records. Feed that is outside the ranges set fur the above mentioned criteria will not be utilized by the testing facility.

4. Water

Municipal tap water following treatment by reverse osmosis will be available ad libitum throughout the study. The purified water will be supplied by an automatic watering system. Monitoring of the drinkin!: water for contaminants will be conducted annually by’ the testing laboratory and the records will be available for inspection. Levels o-f contaminants which may be present are not expected to compromise the purpose of the study. The water meets the standards specified under the EPA National Drinking Water Regulations (40 CFR Part 141).

F. Acclimation

m i

r? 4x- ,

Upon receipt, the animals will be removed randomly from the shipping cartons, examined by qualified personnel, identified with plastic ear tags, and then acclimated to the laboratory conditions for a minimum of 5 days. The animals will be observed daiiy for oved physical or bkhavioral abnormalitia;s, general health/moribundity and mortality.

‘G. Animal Selection

At least 42 animals will be weighed and examined in detail for adverse clinical signs. Animals determined to be suitable as test subjects will be assigned randomly to groups. The animal numbers and the respective body weight


values will be entered into the computer. Homogeneity of groups by weight will be the criteria of acceptance of the randomization. Disposition of animals not selected for study will be documented in the study records.

VI. EXPERIMENTAL DESIGN AND PROCEDURES

A. Study Group Design

This study will consist of a Main Study and a PCA assay:

Main Study Group No. No. of Test

Animals Administration

1 10 N8# 6312322 (Mechl trans.) 5713~“I (10%)

2 10 NB# 6312323 (Mechl non-transf 5713C-K (10%)

3 10 NB#6312331 (Mech3 non-trans) 5713C-M (10%)

4 -10 Control Diet 5713C-H

PCA Assay No. of Test Group No.* Animals’ Administration*

*To be determined and added by protocol amendment.

-

B. Rationale for Dosage Levet Selection

Levels chosen were considered acceptable for determination of titer to the antigen administered based on preliminary work.

C. Dosing Procedures (Main Study)

I. Dietary Dosing (Groups 1-5)

Each group of Main Study Test animals wili be fed the appropriate premade diet as received from the Sponsor ad iibitum from day 0 through day 60 (;: 2 d.ays).

h

-


(44)

Page IO of 19

jy

ii.. I

a


The animals will be checked for general health/mortality and moribundity twice daily during the study. in addition, cage-side observations will be performed once daily and detailed clinical observations will be performed weekly. The detailed weekly observations will include, but not be limited to, evaluations of the skin and fur; eyes and mucous membranes; respiratory, circulatory, autonomic and central nervous system; somatomotor activity and behavioral pattern. Particular attention will be directed to observations of tremors, convulsions, lethargy, other signs of central nervous system depression, salivation and diarrhea.

3. Body Weights

individual body weights will be obtained for ail Main Study test animals (Groups I, 2,3 and 4) on day 0,7,14,21,28,35,42,49 and 56’and prior to blood collection (termination).

4. Food Consumption

Individual food consumption will be measured at least weekly (or more frequently as determined by the Sponsor) on the same day as body weights. Food consumption may be reported as g/animal/day.

5. Collection of Blood Samples

On day 60 (+ 2 days), Main Study test animals (Groups I, 2 ,3 and 4) wilt be anesthetized with isoflurane and blood samples will be obtained intracardially (approximately 2 mL or as much as possible up to 1 Omt- terminal blood collection). Blood collected for serum separation will be allowed to clot. Serum will be haNested according to the Standard Operating Procedures of the Test Facility for preparation of serum specimens. Each serum sample for PCA will be heat inactivated at approximately 56°C for 30 minutes. Approximately 200 UL or up to one- half of each of the serum samples will be collected for return to the Sponsor for possible analysis. All serum samples will be stored frozen (with minimal freeze-thaw if possible).

SLI Study No. 3044,697

6. Unscheduled Deaths and Euthanasia

Page 11 of19

Any animals dying or euthanized moribund during the study wilt be 1 Jr necropsied, The animals will be euthanized by carbon dioxide inhalation

or an intraperitoneal injection of sodium pentobarbitai. Body cavities (cranial, thoracic, abdominal and pelvic) will be opened and examined. No tissues will be retained.

7. Scheduled Euthanasia

immediately following the blood collection, ail surviving animals will be euthanized by carbon dioxide inhalation or an intraperitoneal injection of sodium pentobarbital. A gross necropsy examination will be conducted for surviving animals at the discretion of the Sponsor (e.g., adverse clinical observations) in which case limited tissues may be collected and retained in 10% buffered formaiin for possible future evaluation.

D. PCA Study Procedures

-.

1. Preliminary Procedures

Within 14 days of Main Study blood collection, the animals will be ciipped using a No. 40 size blade from the neck to the rump and down both sides onto the abdomen. When clipping, care will be taken not to abrade the skin. in addition, the back of th’e animals may be depilated: Neet Hair Remover or other appropriate depilatory agent will be placed on the test sites and surrounding areas and left on for a sufficient time to remove hair but no more than fifteen minutes. The depilatory will then be thoroughly removed with a stream of warm water. The animals will be dried with a towel. Test sites will be delineated on the day prior to dosing. The animals will be individually weighed on the day prior to dosing. Note: PCA assay may be conducted by splitting groups and dosing over more than one day if needed, depending upon the final number of PCA animals required.

2. Serum Preparation - Dilutions h

On the day of dosing, serum samples from the Main Study animals will be allowed to thaw at less than 37°C. Each serum tube will be gently mixed prior to removing the sample aliquots. Samples (0.5 mUserum sample or less, if possible) from each of ten animals in each Main Study group

(46)


(l+,vNill be diluted individually for ID dosing. Dilutions of the serum will be made’ by mixing measured aliquots of the sera with measured volumes of sterile, saline to provide the final ratios. The following dilutions will be utilized for d&sing:

Group Dilution’ Dilyent

Group 1 Sterile Saline




‘To be determined and added by. protocol amendment. I

Any change in dilutions requested for use by the Sponsor will be documented in the’raw data and presented in the final report. All dilutions will be used on the day that they are prepared, and the time between preparation and injection wilt be minimized as much as possible,

3. tntradermat Injection Sites

On the day of dosing, animals selected for the PCA Study will be injected intradermally with up to 24 injections. lntradermai injection sites and injections will be determined and added by protocol amendment.

Changes to the dosing regime may be made by the Sponsor not to exceed 24 injection sites. The final dosing regime will be documented in the raw data.

Each injection site area may be re-identified with indelibie ink. In addition, a PCA assay record will be used to identify each PCA test animal by identification number and will specify which individual serum samples are tested on that animal and the location (left dorsal surface or right dorsal surface). In each case, the test sera will be injected in cIose proximity to the shoulder and each successive dilution will be injected in a more posterior position.

A 1 cc tuberculin syringe and an attached 26 or 27 gauge intradermai needle will be utilized.


(47)

Page 13 of 19

4. PCA Challenge

Approximately four hours (5 30 minutes) after the intradermal injections, a chaftenge dose using 1 mL of the appropriate PCA challenge solution (note: an additional ? mL of PCA solution may be injected- if needed) per animal will be injected intracardially using a 3 cc disposable syringe and a 22 gauge, 3/4 or 1 inch needle (or other appropriate size). The animals will be lightly anesthetized with isoflurane to expedite handling at chaltenge. Note: Histamine and Sterile Saline (if utilized) will be injected intradermally approximately 15 minutes after PCA challenge.

5. Challenge Evaluation

Approximately twenty minutes (f 15 minutes) after PCA challenge (intracardial injection of compounds and dye), the animais will be euthanized by carbon dioxide inhalation or an intraperitoneal injection of sodium pentobarbital. The skin will be removed, inverted, photographed (with e.g. a clear ruler) and examined for areas of bluing (the diameter of which will be measured), In addition, the thoracic cavity of each PCA animal will be examined specifically to observe heart and lungs. Also, it will be noted if the musculature of the back is a bluish discoloration.


Any unusual observations, and mortality will be recorded. The animals will be observed for general health/mortality twice daily (once in the morning and once in the afternoon).

7. Body Weights

Individual body weights will be obtained for all PCA Study animals on the day prior to dosing.

8. Unscheduled Deaths and Euthanasia

Any animals dying or euthanized moribund during the study will be necropsied. The animals will be euthanized by carbon dioxide inhalation or an intraperitoneal injection of sodium pentobarbital. Body cavities (cranial, thoracic, abdominal and pelvic) will be opened and examined. No tissues will be retained.

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SLI Study No. 3044.697 Page 14 of 19

VII. PROTOCOL AMENDMENT

Alterations to t.his protocol may be made as the study progresses. No changes in the protocol will be made without the specific consent of the Sponsor’s Representative. ‘A protocol amendment will be prepared and signed by the Study Director, St! Quality Assurance and Sponsor’s Representative for any such changes.

Vlll. DATA REPORTING

One unbound copy of the draft report (if requested) and three copies of the final report (two hound and one unbound) will be submitted to the Sponsor. Thk final report will include all information necessary to provide a complete and accurate description and evaluation of the experimental procedures and results.

The report will include at’least the following information and data:

- Table of Contents - Regulatory Comptiance - Summary - Introduction - Expkimental Design and Test Procedures - Presentation and Discussion of ‘Results - Conclusion - References - Data Tables - Summary of Titers and Diameter of Responses - Summary of Body Weights - Summary of Body Weight Changes - Summary of Food Consumption (g/animal/day) - lndividuai Titers and Diameter of Responses - Individual Clinical Signs - Individual Body Weights - Individual Body Weight Changes - Individual Food Consumption (g/animal/day)

The following additional information may be presented in the appendices: protocol and any amendments, sponsor analytical chemistry report and personnel responsibilities.

P ’ i ,’

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SLI Study No. 3044,697 Page.15 of 19

IX. ANALYSIS OF DATA

The last intradermal injection site on the PCA animal to show a positive response (bluing) may b.e considered to be the titer of that serum sample. If the highest dilution of serum tested demonstrates a positive response, a higher dilution series of that sample may be tested (e.g., 2 or 3 dilutions higher than previously tested). The response wilt be recorded for each dilution. Comparisons of diameters of bluing may also be made.

Statistical analyses will be performed using MicroVax 3100 computer. The level of significance will be a minimum of 5% and ‘testswill be two-tailed. The summary tables will indicate the level of significance detected. The control‘ group data will be compared to the treated data using all groups or by a group by group comparison depending on the test. Data in&ding body weights, weight gain, food consumption will be anaiyzed by one way analysis of variance. if significance is detected, a group by group comparison will proceed with Tukey-Kramer. .

X. MAINTENANCE OF RAW DATA, RECORDS AND SPECIMENS

Ail original data, magnetically encoded records, photographs, specimens and reports from this study are the property of the Sponsor. These materials shall be available at SLI to facilitate auditing of the study during its progress and prior to acceptance of the finai report. All original paper data, the final report, magnetically encoded records, and any specimens will be transferred to the SLI archives for a period of 7 years. The Sponsor ,will be contacted prior to the final disposition of these items.

Xl. REGULATORY COMPLIANCE

A. Guidelines

This study may be submitted to and will be performed in general compliance with the U.S. EPA and FDA Guidelines.

El. Good Laboratory Practices

This study will be performed in general compliance with the principles of the Good Laboratory Practice Standards as described by the EPA (40 CFR Partl60) and FDA (21 CFR Part 58).

F & / : ”

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X11. QUALITY ASSURANCE

The study will be inspected at least once during the in-life phase by the Springborn Laboratories,, Inc., Quality Assurance Unit to assure compliance with Good Laboratory Practice Standards, SLl’s Standard Operating Procedures and for conformance with the protocol and protocol amendments. The final report will be audited prior to submission to the Sponsor to ensure that it completely and ’ accurately describes the test procedures and results of the study.

Xltl. USDA ANIMAL WELFARE COMPLIANCE STATEMENT

This study will comply with all applicable sections of the Final Rules of the Animal Welfare Act regulations (9 CFR) and the Public Health Service Policy on Humane Care and Use of Laboratory Animals (OPRR, NIH, 1986). Wherever possible, procedures used in this study have been designed to avoid or r&nimize discomfort, distress and pain to animais. All methods are described in this study protocoi or in written laboratory standard operating procedures. These procedures are based on the most currently available technologies concerning proper laboratory animal use and management. Methods of euthanasia used during the study are in conformance with the above referenced regulations and the American Veterinary Medical Association Panel on Euthanasia [3]. This study design has been reviewed and approved by Springborn laboratories, Inc. institutional Animal Care and Use Committee (IACUC) for a maximum of 150 animals.

This study is being conducted to evaluate the potential immunological effect of the test article and is not expected to produce undue pan or distress in the animals. Following dosing, the Study Director will be notified by the technician if severe locai reactions occur, if the animals exhibit overt ctinical indications of pain/distress post- dose or if delayed severe skin or clinical changes subsequently develop. If severe responses are noted, the Study Director will contact the Facility Veterinarian and Sponsor to consider an appropriate course of action, In the event that the Sponsor cannot be contacted, the Study Director and/or Facility Veterinarian may authorize treatment or euthanasia of the animais.

XIV. DECLARATION OF INTENT

This study will be listed on the SLJ Quality Assurance Master Schedule for the U.S. Environmental Protection Agency and US Food and Drug Administration.

SLI Study No. 3044697 Page 17 of 19

X\/. PROTOCOt APPROVAL

The Sponsor’s signature below dceumants‘far the S!udy Dlmctot that there are no generatly accapted non-animal a)ternatiuea for this study, and that this study does no0 unnecessarily duplic& my previous expeilmeflts.

Manager of Acute Toxieokgy (SLI)

Vice President and ivfanaging Diracfor (Sll)

,-

Monsanto Company s?udy Monjtor

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XVI. REFERENCES

Page 18 of 19

1. Guide for the Care and Use of Laboratory Animals, DHHS Pubkation No. (NIH) 96-03, 1996.

2. 1993 Report of the American Veterinary Medical Assoc. Panel on Euthanasia, JAVMA, Vol. 202, No. 2, pp. 229-249, January 15, 1993.

(53)


PROTOCOL APPENDIX A

-.

Figure I*

Intradermal Injection Sites

*To be determined and added by protocol amendment.

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SLI Study No. 3044.697 Monsanto Study No. SB-98-212

A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PlGS

PROTOCOL AMENDMENT NO. 1

I) PART TO BE CHANGED/REVISED (EFFECTIVE NOVEMBER 30,1998 AND SEPTEMBER 10, 1998):

I. Purpose

CHANGE/REVISION:

Change the first sentence to read as follows:

Page1 of12

This study was performed to provide an assessment of the allergenic activity of transgenic and non-transgenic cottonseed meal in a guinea pig model. Animals were to be sensitized by feeding diets containing cottonseed meal for approximately sixty days, to induce cottonseed protein specific IgG,,, the dominant reaginic antibody of guinea pigs. Antibody titers were to be measured by passive cutaneous anaphyiaxis following transfer of serum from sensitized animals to naive recipients. However, since the results of the main study were considered to be compromised, the Passive Cutaneous Anaphyfaxis Assay was not conducted.

REASON FOR CHANGE/REVISION:

Requested by the Sponsor based on apparent poor nutritional status of the animals fed the test diets (probable inadequate diet and/or palatability issue) confirmed by severe adverse clinical observations (Vitamin C deficiency-like indications) and severe reduction in body weight/body weight gain. In addition, the feed for Group 1 (fed days O-21) was apparently mislabeled control diet (Group 4) from the manufacturer based on the Gene Feed Assay Results,

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Page2of 12

A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUfNEA PIGS

PROTOCOL AMENDMENT NO. -l

2) PART TO BE CHANGED/REVISED (EFFECTIVE SEPTEMBER IO, 1998):

1V.A. Test Articles

CHANGE/REVISION:

Replace Section 1 to read as follows:

I. Main Study

SLI Identification Phase Sponsor Identifications Nos.

Dietary Dosing (Group 1) NB## 6312322 (Mechl trans) 5713C-1 ’ S98.019.3044 (10%)

Dietary Dosing (Group 2) NB# 6312323 (Mechl non-trans) S98.021.3044 5713C-K (10%)

Dietary Dosing (Group 3) NB# 6312331 (Mech3 non-trans) S98.023.3044 5713C-M (10%)

Dietary Dosing (Group 4) ) Control Diet 5713C-l-i 1 V98.003.3044


PCA dosing was omitted as requested by the Sponsor based on reasons stated previously.

F

SLI Study No. 3044.697 Monsanto Study No. SB-98-212 .

Page3of22

A PASSIVE CUTANEOUS ANAPHYLPXIS ASSAY IN GUINEA PIGS


3) PART TO BE CHANGED/REVISED (EFFECTIVE AUGUST 24, 1998):

IV.A.2. Characteristics

CHANGE/REVISION:

On day 28, the subsamples of the bulk feed will be stored at room temperature rather than refrigerated to reduce potential condensation. In addition, the Sponsor evaluated a sample from each box of diet (3 boxes per diet) on site using a Gene Feed Assay to be described in the final report.

r, REASON FOR CHANGWREVISION: I

Requested by the Sponsor.

4) PART TO BE CHANGED/REVISED (EFFECTIVE AUGUST 17, 1998): s” II

lV.6. Analytical/Retention Samples

CHANGE/REVISION:

Replace this section with the following:

An approximate 20 g analytical sample of each lot of test/control diet will be taken on days 7, 14, 27, 28, 35, 42 and at study termination (day 49) and stored at approximately -70°C for possible Sponsor evaluation. An approximate IO g retention sample of each test/control diet will also be collected from each lot of test/control diet and from each subsample. These samples will be stored refrigerated and wilt serve as the retention samples for all studies conducted with these lots of test/control diets utilized.

r”

R!ZASON FOR CHANGE/REVISION:

To specify the sample collection days due to early termination of the study.

(57)

SLI Study No. 3044.697. Monsanto Study No. SB-98-212

Page 4 of 12

A,PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS

PROTOCOL AMENDMENT NO. I

5) PART TO BE CHANGED/REVISED (EFFECTIVE AUGUST 17,1998):

IV-E. Method of Test Article Preparation for the Main Study

CHANGE/REVISION:

Add the following:

On day 21, an approximate 50 g sample of each box of diet (3 boxes/diet) was sent to Ralston Analytical Laboratories, 824 Gratiot, St. Louis, Missouri, 63102 for analysis.

REASON FOR CHANGE/REVISION: ” ’

Due to significant adverse clinical observations/reduced body weight, the feed was analyzed to determine any feed deficiencies.

6) PART TO BE CHANGED/REVISED (EFFECTIVE AUGUST 26, I 998):

IV.E.3. Food Effective

CHANGE/REVISION:

Change the seventh sentence to the following:

These dietary analyses will be maintained by the Sponsor.


The Sponsor will present these analyses under a separate cover.

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Page 5 of 12




lV.E.4. Water

CHANGE/REVISION:

Add the following to this section:

The purified water will be supplied by an automatic watering system (days O-29) or reverse osmosis water supplemented with 200 mg/L ascorbic acid (Vitamin C) in water bottles (days 30-49).

REASON FOR CHANGUREVISION:

Requested by the Sponsor.


VI.A. Study Group Design

CHANGE/REVISION:

Replace the table as follows:

l’$r~f~~y No. of Test Administration Animals (Days O-21 and Days 28-49)

1 10 NE3# 6312322 (Mechl trans) 5713C-I (10%)

2 10 N&Y 6312323 (Mechq non-trans) 5713C-K (10%)

3 ?O NES# 6312331 (Mech3 non-trans) 5713C-M (10%)

4 10 Control Diet 5713C-Ii

Ma;” p” t;; y N;n yo.;s t Administration (Days 35 - 44 only)

5* 4 NEW 6312322 (Mechl trans) 5713C-I (lo%)**

6’ 4 Control Diet 5713C-I-i

*No Vitamin C supplement; fed/observed at the same time inter&s as in the main study. **Correct diet

SLI Study No. 3044.697 Monsanto Study No. S8-98-212

Page6002

A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN WIMEA PIGS

PROTOCOL AMETWMENT NO. 1


Since there were severe adverse clinical observations/reduced body weight in the main study, any results from the serum of these animals would be considered compromisedlunreliable, a PCA study was not performed. In addition, the feed for Group 1 (fed days O-21) was apparently control diet (Group 4) mislabeled by the manufacturer based on the Gene Feed Assay results. Also, in order to confirm the effects of the diets, “normal stock animals” were fed the specified diets to observe/confirm the onset of adverse effects of this test diet.

9) FART TO BE CHANGED/REVISED (EFFECTIVE AUGUST 17,24 AND 26, 1998):

VI.C.l. Dietary Dosing (Groups I-4)

CHANGE/REVISION:

Replace to read as follows:

Each group of Main Study test animals will be fed the appropriate pre-made diet as received from the manufacturer ad libitum from days 0 -21 and from days 28-49. Aiso on day 28, remove Animal No. 4648 from study. In addition, on day 30, reverse osmosis water suppiemented with 200 mg/L of ascorbic acid (Vitamin C), was provided to the animals (changing water bottles -3 days per week, disconnecting the automatic watering system and refrigerating remaining preparation for use).

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The test diet was stopped, restarted, then the study terminated early for reasons discussed previously. Due to severe adverse clinical observations persisting, the referenced animal was removed from study. In addition, Vitamin C was supplemented in an attempt to reduce Vitamin C deficiency-like signs.

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Page7of?2

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i? PROTOCOL AMENDMENT NO. 1 i 8 b-r

10) PART TO BE CHANGED/REVISED (EFFECTIVE AUGUST 13 and 20,1998):

F k 1 Vl.C.3. Body Weights

yl CHANGE/REVISION:

LJ Replace this section to read as follows:

Individual body weights will be obtained for all.Main Study test animals (Groups I, 2, 3 and 4) on days 0, 7, 14, 17, 21, 24, 28, 35, 38, 42 and 49 (study termination). Animals found dead after day 0 or euthanized moribund will also be weighed .

Fllr

t 1 REASON FOR CHANGE/REVISION:

t : Additional body weights were conducted to determine animal health status due to significant adverse clinical observations. Also, in addition, the study was terminated early.

f? II) PART TO BE CHANGED/REVISED (EFFECTIVE AUGUST 17, 1998 AND 1 1 k 1 EFFECTIVE NOVEMBER 30, 1998):

pl 1 I i Vt.C.4. Food Consumption

CHANGE/REVISION:

Replace the first sentence of this section as follows:

individual food consumption will be measured three times a week. In addition, due to the amount of food spillage on this study, food consumption is not considered to be accurate and therefore will be invalidated.

REASON FOR CHANGUREVISION:

Guinea pigs were fed more frequently than stated in the protocol in order to be fed ad libitum. In addition, food consumption was invalidated as requested by the Sponsor.

Stl Study No. 3044.697 Monsanto Study No. SB-98-212

Page8of+l2 -

A PASSWE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS

PROTOCOL AMENDMENT NO. 1 . . .

12) PART TO BE CHANGED/REVISED (EFFECTIVE AUGUST 17,1998):

VI.C.5. Collection of Blood Samples

CHANGE/REVISION:

Replace as follows:

-

On day 21, due to adverse clinicat observations appearing particularly in Groups 2 and’3 along with significantly reduced weight gain or weight loss, the Sponsor requested the following:

Blood will be collected via the orbital sinus (under light anesthesia) from ail animals along with 5 additional stock animals for comparison. The following parameters will be evaluated:

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A PASSIVE CUTANEOUS ANAPHYLAXlS ASSAY IN GUINEA PIGS

PROTOCOL AMENDMENT NO. 1 ’

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Page9of12

Hematolugy. Parameters Biochemistry Parameters ,

Ery-throcyte Count (RBC) alanine aminotransferase (ALT)

-Iematocrit (Hct) albumin

iemoglobin concentration

Mean Corpuscular hemoglobin concentration :MCHC)

Mean Corpuscular volume

albumin/globulin ratio / 1 alkaline phosphatase

aspartate aminotransferase (AST)

Platelet Count I calcium

Reticuiocyte Count 1 cholesterol

Total Differential Leukocyte Counts blood creatinine I . I.

Mean Corpuscular hemoglobin &O-O I

globulin (calculated)

glucose

electrolytes (Na, K, Cl) .__I

. . phosphorus

1 1 total bilirubin

I total serum protein I 1

1 Urea protein-Urea Nitrogen (BUN)

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-.

1

-I


Serum was not needed for PCA as discussed previously. In addition, in order to better evaluate main study animals’ health, clinical pathology was conducted on all main study animals and 5 stock animals as requested by the Sponsor.

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13)

.

l

14)

Page 10 of 12

A PASSIVE CUTANEOUS ANAPHYWIS ASSAY IN GUINEA PIGS


PART TO BE CHANGEDIREVlSED (EFFECTIVE AUGUST 26,1998):

Vl.C.6. Unscheduled Deaths and Euthanasia

CHANGE/REVISION: *

Replace the last sentence as follows.

On day 30, Animal No. 4648 was euthanized moribund and a gross necropsy examination was performed. In addition, the proximal and distal ends of the ‘femur a2d tibia were collected and the bones processed/decalcified and e{&~ate&.by%.~thoiogist.

fvisbn, &&$&,ANGE/REVISICN:

Requested by the Sponsor in order to determine abnormalities, including growth plate separation (poss‘ibly Vitamin C deficiency-related).

PART TO BE CHANGED/REVISED (EFFECTIVE SEPTEMBER 10,1998) :

VI.C.7. Scheduled Euthanasia

CHANGUREVISICN:

Replace with the following:

-

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c

All animals surviving to study termination (day 49) will be euthanized by carbon dioxide inhalation .or intraperitoneal injection of sodium pentobarbital and discarded without a gross necropsy.

REZASON FOR CHANGUREVISION:

As discussed previously, the study was terminated early. The Sponsor requeste,d that surviving animals be discarded with out necropsy.

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r? i s e 3


Page 11 of 12

r i,


F t PROTOCOL AMENDMENT NO. 1 u 1

IS) PART TO BE CHANGED/REVISED (EFFECTIVE SEPTEMBER IO, 7998):

M.D. PCA Study Procedures

m P * CHANGE/REVISION: F -, _ larsd

r-i Omit this section.

L

* : REASON, FOR CHANGE/REVISION:

Based on the compromised results of the main study, PCA was not required as requested by the Sponsor.

r; 16) PART TO BE CHANGED/REVISED (EFFECTIVE SEPTEMBER IO, 1998):

IX. Analysis of Data

CHANGtiREViSION:

Omit the first paragraph.

m i :

REASON FOR CHANGEIREi/lSION:

* Since no PCA data was collected, this paragraph is not needed.

17) PART TO BE CHANGED/REVISED (EFFECTIVE SEPTEMBER IO, 1998):

Protocol Appendix A

CHANGE/REVISION:

Omit Figure 1.

F” ’ k : REASON FOR CHANGE/REVtSION: I “)S

Since no PCA was conducted, this Appendix is not needed.

SW Study Na. 3W,S%7 Mwsanto Study No. 58-98-212

Page 12 of 12 -

PROTOCdL AiMENDMENT NQ. 1 8 -

Span&s Rapktxekatiwo

Mensanta Company study Monitor

SLI Study No. 3044.697 (66)

APPENDIX B

Results of Feed Gene Assay

SLI Study No. 3044.697 (67)

-

FEED GENE ASSAY

A gene check assay was performed at Springborn Laboratories, Inc., Spencerville, Ohio, by Richard Goodman, Monsanto Company on September 23, 1998 on the boxes of test diet listed in the results below. Apprcximately 5 pellets from each box were removed and placed in test tubes. The samples were then q.s. to approximately 4.0 ml with sterile saline and mixed (vortexed). The samples were allowed to rest for approximately 10 minutes. The subsequent supernate was then transferred to pre-labeled 1.5 ML tubes using sterile, disposable pipettes. A gene check immunoassay testing strip was then placed in each tube. The strips’ were allowed to develop for approximately IO minutes. The strips were

-

-

- then photographed (which remain in the raw data).

The results of the test are as foilows:

Sample Box # Springborn I.D. Purina Lot No. Results

5713C-H 1 V98.003.3044 9848-l Negative” 2 V98.003.3044 9848-l Negative 3 V98.003.3044 9848- 1 Positive ’

5713c-I 1 598.019.3044 9848-2 Positive 2 S98.019.3044 9848-2 Negative 3 S98.019.3044 9848-2 Positive

5713C-K I S98.021.3044 9848-4 Negative 2 S98.021.3044 9848-4 Negative 3 S98.021.3044 9848-4 Negative

5713C-M 1 S98.023.3044 9848-6 Negative 2 S98.023.3044 9848-6 Negative 3 S98.023.3044 9848-6 Negative

“Approximately eight minutes later, a second test strip was read and appeared to be positive for the BT gene; upon review of the first strip, what appeared originally as an imperfection was now clearly positive, but weak (light pink).

.%-

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APPENDIX C

Certificate of Analysis (Provided by the Manufacturer)

(69) ST2 Stxdy No. 3044.697

clfrz;M,fi

3050 Spruce Streef Saint Louis, Missouri 63103 USA

T&phone 800-521-8956 l (314) 771-5765 Fax 800-325-5052 l (314) 7715757

emailr sig-aldOsial.com http:llwww.sigma.srai.com

SPRINGBORN LABS 640 N ELIZABETH ST SPENCERVILLE OH 45887

C-ERTIFlCATE OF ANALYSIS ASCORBIC ACID, APPROX. -325 MESH USP XXIII PRODUCT NO: A2343 . CAS NO: 50-81-7 LOT NO: 096803631 FORMULA: C6H806 FORMULA WEIGHT: 176.13 STORE AT ROOM TEMPERATURE

-

-

TEST SPECIFICATSON RESULTS -

IDENTITY PASS PASS -

SPECIFIC ROTATION

f20.5 DEG TO i-21.5 DEG +20.9 DEG

RESIDUE ON IGNITION

NMT 0.1% PASS

HEAVY METALS NMT 0.002% PASS

- ORGANIC VOLATILE METHOD IV TESTING IMPURITIES .-

I ASSAY 99.0% TO 100.5% 99.8% - I

NOTE ALL RESULTS SUPPLIER DATA _ I I

QC:-ACCEPTANCE DATE MAY 1997

DAVID FELDKER MANAGER, ANALYTICAL SERVICES

238/980825#l/SMLI DOCUMENT DATE: 08/25/98

SIGMA-ALDRICH, INC. UARRANTS THAT ITS PRCDUCTS CONFORM TO THE INFORMAT 1 CONTAINED IN THIS AND OTHER STGMA-ALDRICH

PUBLJCATICNS. PURCHASER MUST DETERMINE THE SUITABILITY OF THE PRCWCT( :FOR THElR PARTTCULAR USE. ADDITIONAL TERMS I AND CONDITIONS MAY APPLY. PLEASE SEE REVERSE SIDE OF THE INVOICE OR PA NG SLIP.

SIGMA BRAND PRODUCTS ARE SOLD EXCLUSIVELY THRCUG

SLI Study No. 3044.697 (70)

APPENDIX D

Clinical Pathology Methodology


I.

1.

2.

3.

4.

5.

6.

(71)

A. Blood Collection

Blood c&ction. Blood samples for hematology were collected into tubes containing I1(,EDTA anticoagulant. Blood samples for biochemistry were collected into tubes containing no anticoagulant.

B. Hematology

Red blood cell (RBC) and white blood ceil (WBC). A Coulter S Plus IV hematology analyzer is used in determining the RBC and WBC. A Coulter S-Cal kit is used to calibrate the instrument and Coulter T&Pack control sera are used in monitoring accuracy and precision of the instrument.

Hemoglobin (HGB). Hemoglobin is measured on the Coulter S Plus’ IV hematology analyzer. A iysing reagent lyses the RBC and converts the released HGB to a stable cyanide-containing pigment which is measured photometrically. Coulter Tri-Pack control sera are used in monitoring accuracy and the precision of the instrument.

Mean corpuscular (erythrocyte) volume (MCV). The MCV is measured on the Coulter S Plus IV hematology analyzer. Coulter T&Pack control sera are used in monitoring the precision and accuracy of the instrument.

Mean corpuscular (erythrocyte) hemogtobin (MCH). The MCH is computed electronically on the Coulter S Plus IV hematology analyzer using the following equation:

MCH (pug) = 10 x HGB RBC

Mean corpuscular (erythrocyte) hemoglobin concentration (MCHC). MCHC is computed electronically on the Coulter S Plus IV hematology analyzer using the following equation:

MCHC (g/dt) = 100 x HGB HCT

Hematocrit (HCT). Hematocrit is the relative volume of erythrocytes and is computed electronically on the Coulter S Plus IV hematology analyzer using the following equation:

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SLI Study No. 3044.697 (72)

HCT (%) = RBC x MCV IO

7. Piatelet count (PLT). The platelet count is determined on the Coulter S Pius IV hematology analyzer. Coulter 4C Tri-Pack control sera are used to monitor precision and accuracy of the instrument.

8. Differential leukocyte and nucleated red blood cell counts (NRBC). A Bayer Hema-tek slide stainer is used to automatically stain the slides using a combination of Wright stain, buffers and rinse solutions wright-Giemsa stain). One hundred white cells are counted and identified under oil immersion (100x) using the Nikon Labophot microscope. The slides are also checked for any abnormal RBC morphology. Nucleated red blood cell (NRBC) counts are counted as per 100 leukocytes. If the nucleated red blood cell count exceeds five or more in the differential count, then the WBC count from the Coulter S Plus IVmust be corrected by using the following equation:

100 200 + Number of NRBC x WBC count = corrected WBC count

a_ i _, I.( . 9. Reticulocyte count using the Miller Disc Reticle method. Equal amounts of

&EDTA blood and new methylene blue N solution are mixed, incubated and spread on a microscope slide. A light microscope with a 100x objective and a ‘IOx ocular fitted with a mitler disc reticle is used to enumerate reticulocytes and red blood cells. Approximately 300 cells are counted and the following equation is used:

% Reticulocyte = number of reticulocvtes x 100% number of erythrocytes x 9

D. Biochemistry

All biochemistry parameters are performed on the Beckman Synchron CX-5 chemistry analyzer. Synchron Control Level I and Level II are used as control sera to monitor accuracy and precision of the instrument. Listed below are the biochemistry parameters and their methodoiogy.

1. Albumin (ALB). Beckman Albumin reagent is used to measure the albumin ’ concentrat.ion by a timed-endpoint method. In the reaction, the albumin combines with Bromcresoi Green (BCG) to form a colored product. The system monitors the change in absorbance at 600 nanometers at a fixed-time intervai. This change in absorbance is directly proportional to the

.

St1 Study No. 3044.697 (73)

2.

3.

concentration of albumin in the sample and is used by the Synchron CX-5 System to calculate and express the albumin concentration.

.Alkaline phosphatase (ALP). Beckman ALP Reagent is used to measure the ALP activity by an enzymatic rate method using a 2-Amino-2-Methyl-l -Propanol (AMP) buffer. In ?he reaction, the ALP catalyzes the hydrolysis of the colorless organic phosphate ester substrate, p-Nitrophenyiphosphate, to the yellow colored product, p-Nitrophenol, and phosphate. This reaction occurs at an alkaline pH of 10.3. The system monitors the rate of change in absorbance at 410 nanometers over a fixed-time interval. This rate of change in absorbance is directly proportional to the activity of ALP in the sampte and is used by the Synchron CX-5 System to calculate and express the ALP activity.

Alanine aminotransferase (ALT’). Beckman ALT Reagent is used to measure the ALT activity by an enzymatic rate method. In the reaction, the ALT catalyzes the reversible transamination of L-alanine and alpha-ketoglutarate to pyruvate and L-glutamate. The pyruvate is then reduced to lactate in the. presence of lactate dehydrogenase (LDH) with the concurrent oxidation of b-Nicotinamide Adenine Dinucleotide (reduced form) (NADH) to b-Nicotinamide Adenine Dinucleotide (NAD). The system monitors the rate of change in absorbance at 340 nanometers over a fixed-time interval. This rate bf change in absorbance is directly proportional to the activity of ALT in the sample and is used by the Synchron CX-5 System to calculate and express the ALT activity.

4. Aspartate aminotransferase (AST). Beckman AST Reagent is used to measure the AST activity by an enzymatic rate method. In the reaction, the AST catalyzes the reversible transamination of L-aspartate and alpha- ketoglutarate to oxaloacetate and L-gtutamate. The oxaloacetate is then reduced to malate in the presence of malate dehydrogenase (MDH) with the concurrent oxidation of b-Nicotinamide Adenine Dinucleotide (reduced form) (NADH) to b-Nicotinamide Adenine Dinudeotide (NAD), The system monitors the rate of change in absorbance at 340 nanometers over a fixed-time interval. This rate of change in absorbance is directly proportional to the activity of AST in the sample and is used by the Synchron CX-5 System to calculate and express the AST activity.

5. Calcium (CA). Beckman Calcium Reagent is used to measure the calcium concentration by a timed-endpoint method. ln the reaction, the calcium combines with Arsenazo III to form a colored product. The system monitors the change in absorbance at 650 nanometers at a fixed-time interval. This change in absorbance is directly proportional to the concentration of calcium

-

-

-

-

-

-

-

r I :

Stl Study No. 3044.697 (74)

r

: .!

F”: b , * 1

6.

7.

in the sample and is used by the Synchron CX-5 System to calculate and express the calcium concentration.

Cholesterol (CHOL). Beckman Cholesterol Reagent is used to measure the cholesterol concentration by a timed-endpoint method. In the reaction, the cholesterol esterase (CE) hydrolyzes cholesterol esters to free cholesterol and fatty acids. The free cholesterol is oxidized to cholesten-3-one and hydrogen peroxide by cholesterol oxidase (CO). Peroxidase catalyzes the*reaction of hydrogen peroxide with 4-aminoantipyrine (Q-AAP) and phenol to produce a colored quinoneimine . product. The system monitors the change in absorbance at 520 nanometers at a fixed-time interval. This change in absorbance is directly proportional to the concentration of cholesterol in the sample and is used by the Synchron CX-5 System to calculate and express the cholesterol concentration.

Creatinine (CREA). Beckman Creatinine Reagent is used to measure the creatinine concentration by a modified rate Jaffe method. In the reaction, the creatinine combines with picrate in an alkaline solution to form a creatinine-picrate complex. The system monitors the rate of ‘change in absorbance at 520 nanometers over a fixed-time interval. This rate of change in absorbance is directly proportional to the concentration of creatinine in the sample and is used by the Synchron CX-5 System to calculate and express the creatinine concentration.

8. Glucose (GLU). Beckman Glucose Reagent is used to measure the glucose concentration by a timed-endpoint method. In the reaction, hexokinase (HK) catalyzes the transfer of a phosphate group from adenosine triphosphate (ATP) to glucose to form adenosine diphosphate (ADP) and glucose-6-phosphate. The glucose-6-phosphate is then oxidized to 6phosphogluconate with the concomitant reduction of b-Nicotinamide Adenine Dinucleotide (NAD) to b-Nicotinamide Adenine Dinucleotide (reduced form) (NADH) by the catalytic action of’glucose-6-phosphate dehydrogenase (GGPDH). The system monitors the change in absorbance at 340 nanometers at a fixed-time interval. This change in absorbance is directly proportional to the concentration of glucose in the sample and is used by the Synchron CX-5 System to calculate and express the glucose concentration.

9. Phosphorus (PHOS). Beckman Phosphorus Reagent is used to measure the phosphorus concentration by a timed-endpoint method. In the reaction, inorganic phosphorus reacts with ammonium molybdate in an acidic solution to form a colored phosphomolybdate complex. The system monitors the change in absorbance at 330 nanometers at a fixed-time interval. This change in absorbance is directly proportional to the concentration of

SLI Study No. 3044.697 (75)

phosphorus in the sample and is used by the Synchron CX-5 System to calculate and express the phosphorus concentration.

10. Total bilirubin (TBIL). Beckman Total Bilirubin Reagent is used to measure the total bilirubin concentration by a timed-endpoint Diazo method. In the reaction, the biiirubin reacts with diazo reagent in the presence of caffeine, benzoate, and acetate as accelerators to form azobilirubin. The system monitors the change in absorbance at 560 nanometers at a fixed-time interval. This change in absorbance is directly proportional to the concentration of total bilirubin in the sample and is used by the Synchron CX-5 System to calculate and express the total bilirubin concentration.

11.

12.

Total protein (TP). Beckman Total Protein Reagent is used to measure the total protein concentration by a timed-endpoint biuret method. In the reaction, the peptide bonds in the protein sample bind to cupric ions in an alkaline medium to form colored peptide/copper complexes. The system monitors the change in absorbance of 560 nanometers at a fixed-time interval. This change in absorbance is directly proportional to the concentration of total protein in the sample and is used by the Synchron CX-5 System to calculate and express the total protein concentration. -

Sodium (Na), potassium (K), chloride (Cl). The Synchron CX-5 System determines sodium, potassium, and chloride by measuring electrolyte ion activity in solution. Measurements are made by ion selective electrodes. A precise volume of sample is mixed with a buffer solution at a ratio of I:20 to establish a constant ionic strength and to set a constant activity coefficient for the electrodes. With constant activity established, the electrode system is calibrated to concentration values. This mixture is transported to the flow cell which houses the electrodes. Sodium, potassium, and chloride determinations are made by measuring potentials developed at the face of ion-selective electrodes.

-

-

-

-

-

-

13. Globulin (GLB). Globulin parameter is not measured directly but is calculated using the following equation:

Globulin g/dL = Total Protein - Albumin

-

n

SLI Study No. 3044.697 (76)

APPENDIX E

Individual Clinical Observations

SLI STUDY NO.: 3044.697 CLIENT: HONSANTO COHPANY

A PASSIVE CUTANEOtJS ANAPHYLAXIS ASSAY IN GUINEA PIGS INDIVIDUAL CLINICAL OBSERVA'MOpfS

(POSITIVE FINDINGS)

PAGE 1

--_---_____---__I______I__ _________^____c^_I__________I___________---------------------------------------------------- ,2-----

ANIUAL CLINICAL DAY OF 11111111112222~22222 NO. OBSERVATIONS STUDY 01234567890123456789.012345678

--^---_______________________________I__------------------------------- ______^_- __________c_____________________ ----I--

4606 F GROUP 1: 5713C-I (10%) FECAL STAIN P

4610 F -GROUP 1: 5713C-I (10%) FECAL STAIN P

4627 F GROUP 1: 5713C-I (10X) SOFT STOOLS P FECAL STAIN P

4633 F, @OUP 1; 5713C-I (10%) SOFT STOOLS FECAL STAIN

P P P

4642 F GROUP 1: 5713C-1 (10X) DECREASED FOOD CONSUHPTION P

4643 F GROUP 1: 5713C-1 (10%) FECAL STAIN P

4649 F GROUP 1: 5713C-I (10%) FEW FECES PP SOFT STOOLS P FECAL STAIN P DECREASED FOOD CONSU?lPTION P

__--------------_-_______^______ ------____-___________I_________________--------------------------------------- _^.._---^-c-------- GRADE CODE: l-SLIGHT 2-HODERATE 3-HARKED P-PRESENT

I I I I I I ; I 1 I 1 ‘I sl I 1 I

SLI STUDY NO.: 3044.697 CLIENT: lloNsANI0 COKPANY

A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS INDIVIDUAL CLINICAL OBSERVATIONS

(POSITIVE FINDINGS)

PAGE 2

--------------------___I__^__c I________---------------------------------------------------------------------------------- ------e

ANIHAL CLINICAL DAY OF 1111111111222222222 NO. OBSERVATIONS STUDY 01234567890123456789012345678

------------------------------,,,,-----------------------------------------~-----------------------------------------------

4602 F GROUF 2: 5713C-K (10x) FEU FECES FECES MALL IN SIZE DECREASED FOOD CONSUBPTION

PP< PPPP

P

4608 F GRGUP 2: 5713C-K (10X) FECES SHALL IN SIZE ROUGH COAT

P P

4609 F GROUP 2: 5713C-K (10x) FECES SBALL IN SIZE SOFT STOOLS ROUGH COAT FECAL STAIN DECREASED FOOD CONSUBPTION

PPPPP

'P

PP

4611 F GROUP 2: 5713C-K (10%) FEW FECES P P PPP FECES MALL IN SIZE PPPPPPPPP SOFT STOOLS PP ROUGH COAT PP PPP FECAL STAIN P PP EXTREBITIES APPEAR BLUE IN COLOR PPP DISTENDED ABDDHEN PPP DEBYDRATION PP DECREASED FOOD CONSUMPTION PPP FOUNDDEAD P

---------------___-_------------------------------------------------------------------------------------------------------------- GRADE CODE: L-SLIGHT 2-HODERATE 3-HARKED P-PRESENT

A

2 -

SLI STUDY NO.: 3044.697 CLIENT: HONSrn COBPANY

A PASSIVE CUTANEOUS ANAPBYLAXIS ASSAY IN GUINEA PIGS INDIVIDUAL CLINICAL OBSERVATIONS

(POSITIVE FINDINGS)

PAGE 3

ANIHAL CLINICAL DAY OF 1111111111-222222222 NO. OBSERVATIONS STUDY 01234567890123456789012345678

4617 F GROUP 2: 5713C-K (10%) FEW FECES FECES SHALL IN SIZE SOFT SlnOLS ROUGH COAT DECREASED FOOD CONSUHPTION

P P

P PPPPPP PP

4620 F GROUP 2: 5713C-K (10%) FEW FECES FECES SMALL IN SIZE DISTENDED ABWHEN DECREASEDFOOD CONSUMPTION

4632 F GROUP 2: 5713C-K (10X) FEW FECES FECES SHALL IN SIZE ROUGH COAT DECREASED FOOD CONSUHPTION

PP P PPP PPPPP

PP PPP PPP 2

P P

P P

4639 F GROUP 2: 5713C-K (10%) FEW FECES P FECES SHALL IN SIZE PPPPP ROUGH COAT PPPP DECREASED FOOD CONSUH.PTION PP P

____------------c------------------------------------------------------------------------------------------------- ---'----------- GRADE CODE: l-SLIGHT 2-HODERATE 3-HARKED P-PRESENT

SLI STUDY NO.: 3044.697 CLIENT: HONSANTGCOBPANY

A PASSIVE CUTANF+OUS ANAPHYiMIS ASSAY IN GUINEA FIGS INDIVIDUAL CLINICAL OBSERVATIONS

(POSITIVE FINDINGS)

PAGE 4

c--------------------,,,,,,_____________--------*---------------------------------------------------------------~----------------


------------I---------------------- ----,,,-----,,,-,,--_,___-,,___,,_,_,___-------------------------------------~-~--------- -mm--

4641 F GROUP 2: 5713C-K (10X) FEU FECES FECES SHALL IN SIZE URINESTAIN ROUGH COAT

P P

P PPPP

4645 F GROUP 2: 5713C-K (10x) FEW FECES FECES SHALL IN SIZE ROUGH COAT DECREASED FOOD CONSUBPTION

P PP P P PP

4612 F GROUP 3: 5713C-H (10x) FEW FECES URINE STAIN

P P

4623 F GROUP 3: 5713C-H (10X) FEW FECES FECES SBALL IN SIZE SOFT STOOLS ROUGH COAT

PP

PPP

P P P

4628 F GROUE' 3: 5713C-M (10%) FEW FECES PP FECES SHALL IN SIZE PPP ROUGH COAT PPPPP DECREASED FOOD CONSIJBFTION P

---------------_----_______^______I_____--------------------------------------------------------------------------------- ---em--- GRADE CODE: l-SLIGHT 2-HODERATE 3-BARKED P-PRESENT

-.

SLI STUDY NO. : 3044.697 CLIENTz HONSANTO COMPANY

A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS INDIVIDUAL CLINICAL OBSFRVATIONS

(pOSITIVE FINDINGS)

PAGE 5

---------‘-------c*---------------------------------------------------*---------------------------------------------------------- ANIHAL CLINICAL DA? OF 1111111111222222222

NO. OBSERVATIONS STUDY 01234567890123456789012345678 -----------------*-*____I_______________-----------------------------------------------------------------------------------------

4629 F GROUP 3: 5713C-H (10%) FECES. MALL IN SIZE DECREASED FOOD CONSU’BPTION

PPP P P

4630 F GROUP 3: 5713C-H (10%) FEW FECES FECES SHALL IN SIZE DECREASED FOOD CONSUBE’TION

4646 F GROUP 3: 5713C-H (10%) SALIVATION FECES SHALL IN SIZE SOFT mLs ROUGH COAT FECAL STAIN DISTENDED ABDOHEN

PPP PP

PP .

P P PP P

PPP PPPPPPP PPP

P P P

4648 F GROUP 3: 5713C-H (10%) FEW FECES PPPPPPPP FECES SHALL IN SIZE PPPPPPPPP P SOFT STOOLS PP ROUGH COAT PPPPPg?PPP EXTREHITIES APPEAR BLUE IN COWR PPPPP DISTENDED ABDOHEd PPPPP DEHYDRATION PP DARE HATERIAL AROUND EYE(S) P DECREASED FOOD CONSUMPTION PPP PPP ’ REMOVED FROH STUDY, BUT NOT EUTHANIZEXI AT THIS TIHEa P

-*----*---------**I-____1_1_________^__1---------------------------------------------------------------------------------------- &WE CODE: l-SLIGHT 2-HODERATE 3-BARKED P-PRESENT WlBAL EUTHANIZED ON DAY 30; CLINICAL OBSERVATIONS NOTED AT THAT TME INCLUDED FECES SHALL IN SIZE, SOFT STOOLS, ROUGH COAT, XAL STAIN - SLIGHT, SKIN BLUE IN COLOR - EXTREHITIES, AND DISTENDED ABDOBEN - BACK BONE PROMINENT UPON HANDLING.

SLI STUDY NO.: 3044.697 CLIENT: HoNSrn COHPANY


(POSITIVE FINDINGS)

PAGE 6

---------------------------------^------------------------------------------------------------~----------------------------------- ANIHAL CLINICAL DAY OF 1111111111222222222

NO. ‘OBSERVATIONS STUDY 0123456789012345678901234-5678 --------------------______________I_____---------------------------------------------.--------------- ------------~-^“--“--------“’

4650 F GROUP 3: 5713C-H (10x) FIW FECES FECES SHALL IN SIiE ROUGH COAT

P PP

P

4614 F GROUP 4: 5713C-H FEW FECES FECES SHALL IN SIZE SOFT STOOLS FECAL STAIN

P P

PP PPP

4621 F GROUP 4: 5713C-H SOFT STOOLS

’ URINE STAIN P P

4622 F GROUP 4: 5713C-H FECES SHALL IN SIZE P DECREASD FOOD CONSUHPTION

4625 F GROUP 4: 5713C-H SOFT SmOLS FECAL STAIN

PPP PPP

4636 F GROUP 4: 5713C-H FEW FECES P FECES SHALL IN SIZE P SOFT smoLs P P

--_________________I____________________----------------------------------------------------------------------------------------- GRADE CODE: l-SLIGHT 2-HODERATE 3-HARKED P-PRESENT


A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PI& INDIVIDUAL CLINICAL OBSERVATIONS

(POSITIVE FINDINGS)

PAGE 7 .

--------------------__________^_I_______---------------------------------------------------------------------------------- ----I-- ANIHAL CLINICAL DAY OF 1111111111222222222

NO. OBSEBJlATXONS STUDY 01234567890123456789012345678 --------------------____^__LI__________ ---------------------------------------------------------------------------~.--------------

4638 F GROUP 4: 5713C-H FEW FECES FECES SHAJiL IN SIZE SOFT STQOLS FECAL STAIN

PP PP P

P

4644 F GFtOUP 4: 571$C-H FEW FECES P FECES SHALL IN SIZE P SOFT STOOLS P URINE STAIN P

------^--------_-------- -I--c-----I---__^_-------_--------------------------------------------------------------------------------- GRADE CODE: l-SLIGHT 2-HODERATE 3-HARKED P-PRESENT

SLI STUDY NO.: 3044.697 CLIENT: HGNSANTG COHPANY

A PASSIVE CUTANEOUS ANAPHYIhXIS ASSAY IN GUINEA PIGS PAGE S INDIVIDUAL CLINICAL OBSERVATIONS

(POSITIVE FINDINGS)

--------------------_I__________________------------------------------------------------------------------------------------ -----

ANIHAL CLINICAL DAY OF- 233333333334444444444 NO. OBSERVATIONS STUDY 901234567890123456789 ,

----I-----^-----__--_____ -_--I^-------^--------- ------------,,,-,,,---,,-,-,,,,-,,,,-,-----------------------------------------~--------------

4606 F GROUP 1: 5713C-I (10%) FEW FECES P FECES SHALL IN SIZE P SOFT sTooLs P FECAL STAIN P P DECREASED WATER CONSUHFTION P SCHEDULEDELlTBfQ&SIA

4610 F GROUP 1: 5713C-I (10%) FEW FECES FECES SHALL IN SIZE DECREASED WATER CONSUHPTION SCHEDULED EUTHANASIA

P PP P P

4619 F GROUP 1: 5713C-S (10X) FECES SHALL IN SIZE P FECAL STAIN P SCHEDULED EUTHANASSA

4626 F GROUP 1: 5713C-I (10%) URINE STAlN FECAL STAIN

P.

SCHEDULEDEUTHANASIA

P

P P

P P

4627 F GROUP 1: 5713C-I (10x) FEW FECES PP FECES SKALL IN SIZE PP DECREASEDWATERCONSUttFTION P SCHEDULEDEUTtiANASIA P

--___--_____--^_I_-_____________________-------------------~--------------------------------------------------------------------- GRADE CODE: I-SLIGHT 2-HODERATE 3-d P-PRESENT

[II STUDY NO.: 3044.697 IIENT: MUNSANTO COMPANY

A PASSIVE CUTANEOUS ANhE'HYLAXIS ASSAY lI.l GUINEA PIGS INDIVIDUAL CLINICAL OBSERVATIONS

(POSITIVE FINDINGS)

PAGE 9

--------------------____^____L1_________---------------------------------------------------- __--------------“---_____3_^_ ----^I-

4Nwd.4 CLINICAL DAY OF 233333333334444444444 NO. OBSERVATIONS STUDY 901234567890123456789

--------------L---------------------------------------------------------------------------------- ___----c---------_------ ----..e-

4633 F GROUP 1: 5713C-I (10%) FEW FECES FECES SHALL IN SIZE FECAL STAIN DECREASED FOOD CONSUHF'TION SCHEDULE0 EIlBmNAsrA

PP .P P

P PP

4635 F GROUP 1: 5713C-I (10%) FECES SHALL IN SIZE SCHEDULEDEUIHANASIA

4642 F GROUP 1: 5713C-I (10%) FEW FECES P FECES SHALL IN SIZE P SOFT STOOLS P DECREASED WATER CONSUMPTION P SCHEDULEDEU'IMNASIA

4643 F GROUP 1: 5713C-I (10%) FEW FECES URINE STAIN FECAL STAIN SCHEDULEDEUTHANASIA

.P

4649 F GROUP 1: 5713C-I (10%) URINE STAIN SCHEDULED EUTHANASIA

P

___------I-^-----_--________L___________--------------------------------------------------

ME CODE: l-SLIGHT 2-HODERATE 3-HARKED P-PRESENT

P

P

P P

P

P

P

P P

_---_--L__c_____---_-- -m--e..- ------

SLI STUDY NO.: 3044.697 CLIENT: H@wNTOcoHFANY

A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS INDMDUAL CLINICAL OBSERVATIONS

(POSITIVE FINDINGS)

PAGE 10

,---.m-------s------ -------------.-----‘---- ------------------_-_____c______________------------------------------------------------

ANIHAL cLlMCAL DAY OF NO. OBSERVATIONS

'233333333334444444444 STUDY 901234567890123456789

--------------------____c_____c___I_____--------------------------------------------------~--------------------------------------

4602 F GROUP 2: 5713C-K (1OZ) SCHEDULED ElJlmNAsIA

4608 F GROUP 2: 5713C-K (10%) FEW FECES FECES SHALL IN SIZE SOFT SMOLS URINESTAIN FECAL STAIN DECREASED FOOD CONSUHFTION DECREASED WATER CONSUHPT.ION SCHEDULED EUTHANASIA

4609 F GROUP 2: 5713C-K (10%) SOFT SmoLS ScHEDULEDEUTHANASIA

4617 F GROUP 2: 5713~~K (10x) ROUGH COAT HAIRLOSS SCHEDULEDEUTHANASIA

4620 F GROUP 2: 5713C-K (10%) FEW FECES

P P PPP PPP

P P

P P PP

PPP

P

P PPPP P P FECES SHALL IN SIZE P PPPPPPPPPPPPPPPPPPP SOFT STGOLS P URINE STAIN P P PPPPP ROUGH COAT P P PPP FECAL STAIN P

----------------------------------------------- ___________________-____________________------------------------------------------ GRADE CODE: l-SLIGHT 2-HODERATE 3-HARKED F-FRE;SENT

P

P P

P P P P

P

PPP

P

SLI STUDY NO.: 3044.697 CLIENT: HONSANTG COUPANY


(POSITIVE FINDINGS)

PAGE 11

--------------------__________^_________------------------------------------------------------------------------------------ -----

ANIHAL CLINICAL DAY OF 233333333334444444444 NO. OBSERVATIONS STUDY 9012345678901234567S9

---------------------------------------------------------------------------------------------------------------------------------

4620 F GROUP 2: 5713C-K (10%) (CONTINUED) EXTREMITXES APPEAR BLUE IN COLDR DISTENDED ABDOHEN DECREASED FOOD CGNSUHPTION DECREASED WATER CONSUHPTION SCHEDULED EU'DLANASIA

PPPPPP PPPPPPP

PP PPPP P PPPPPP PPPPPP

P

4632 F GROUP 2: 5713C-K (10%) FECES SHALL IN SIZE ROUGH COAT DISTENDED ABDOHEN DECREASED FOOD CONSUHPTION DECREASED WATER CONSUM'TION SCHEDULED EU'DMASIA

P PPPPPPPPP P P P P

PPPPPP PPPPP

PPP PP PP P

4639 F GROUP 2: 5713C-K (10%) FEW FECES FECES SHALL IN SIZE ROUGH COAT EXTREHITIES APPEAR BLUE IN COLOR DISTENDED ABDOMEN DECREASED FOOD CONSUHPTION DECREASED WATER CONSUMPTION SCHEDULEDEUTHANASIA

----------------------------------------------------- GRADE CODE: l-SLIGHT 24fODFXATE 3-HARKED P-PRESENT

PPPP PP PPPP PPPPPPPPPPPPPP

PPPP PPPPPP PPP

PPPPPP PP P PPP

PPP P P

---------______-_______________________I----------------------------- -------

SLI STUDY NO.: 3044.697 CLIENT: HONSANTG COHPANY

A PASSIVE CUTANEOUS ANAPHYLiiXIS ASSAY IN GUINEA PIGS , INDIVIDUAL CLINICAL OBSERVATIONS

(POSITIVE FINDnuCS)

PAGE 12

--------------------___I_________I______-----------------------------------------------------------------------------------------

ANIHAL CLINICAL DAY OF 233333333334444444444 ; NO. OBSERVATIONS STUDY 901234567890123456789

---------------------------------------------------------------------------------------------------------------------------------

4641 F GROUP 2: 5713C-K (10X) FECES SHALL IN SIZE URINESTAIN DECRF.ASF.D WATER CONSUM?TION SCHEDULED EUTHANASIA

P PP P P P P

P

4645 F GROUP 2: 5713C-K (10%) SCHEDULEDEUTUANASIA P

4612 F GROUP 3: 5713C-Ii (10%) FEW FECES FECES SHALL IN SIZE DECREASED FOOD CONSIJBPTION scHEI)uLED IYJWANASU P

4623 F GROUP 3: 5713C-H (10%) FECES SHALL IN SIZE URINE STAIN SCHEDULED EUlWLNASIA

PP P P

4628 F GROUP 3: 5713C-H (10X) FEW FECES P SCHEDULEDEUDIANASIA P

----------I---------___^_I____^_________----------------------------------------------------------------------------------------- GRADE CODE: l-SLIGHT 2-HODERATE 3-HARKED P-PRESENT

SLI S’IUDY NO.: 3044.697 CLIENT: HONSAKM COHPANY

A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS PAGE 13 INDIVIDUAL CLINICAL OBSERVATIONS

(POSITIVE FINDINGS) ’

------^-___----__---__________________I_---------------------------- -------- -------- --*---- _--m-e _______-_____-I-_I--___c________

ANIHAL CLINICAL DAY OF 2333333333344444444‘4.4 ;’ NO. OBSERVATIONS STUDY 901234567890123456789. m--w- -^--____________________________________------------------- -----c...----------.-- _________________---________I___________------ .

4629 F GROUP 3: 5713C-H (10X) FECES SHALL IN SIZE URINE STAIN SCHEDULED EU’IMNASIA

PP P P

4630 F GROUP 3: 5713C-H (10%) FEW FECES FECES SHALL IN SIZE DECREASED FOOD CONSUHPTION DECREASED WATER CONSUtlPTION SCHEDULED EUTHANASIA

4640 F GROUP 3: 5713C-H (10%) SCHEDULED EUTHANASIA

4646 F GROUP 3: 5713C-H (10%) FECES SHALL IN SIZE SCHEDULED EUllfANASIA

PPP P PP PP

P P

P

P P

4647 F GROUP 3: 5713C-H (10%) FEW FECES P -3 FECES SHALL IN SIZE PP SCHEDULED EUTHANASIA P

___-&^s- _l___-_--l^------l----- -----a- ____---^----.----- ___----- _-_-m-e -------------^-______________^___I______------------------- GRADE CODE: l-SLIGHT 2-HODERATE 3-w P-PRESENT

I I I i I I I

SLI STUDY NO.: 3044.697 CLIENT: KJNSAE-FM) CORPANY


(POSITIVE FMXNGS)

PAGE 14

--------------------___I________________---------------------------------------------------------------------------- _a.^------a----


--------------------____________________-------------------------------------------------- "'--'--'---'-'---^--'---"-------c---------

4650 F GROUP 3: 5713C-H (10X) FEW FECES FECES SHALL IN SIZE SOFT SMOLS ROUGH COAT DECREASED FOOD CONSUHi'TION DECREASED WATER CONSUBPTION SCHEDULED EUTHANASIA

PP PPPPP PP PP PPPPPPPPPP

P PP P P

PP PPPPPP P

P

4601 F GROUP 4: 5713C-H FEW FECES FECES SMALL IN SIZE SOFT STOOLS FECAL STAIN DECREASED FOOD CONSUHFTION SCHEDULED EUTHANASIA

P PP

P P

P P

4604 F GROUP 4: 5713C-H FEQ FECES FECES SMALL IN SIZE DECREASED WATER CONSUBPTION SCHEDULFDEUTHANASIA

PP P . P

P

4614 F GROUP 4: 5713C-H FECES SHALL IN SIZE PPPP FECAL STAIN P P DECREASED FOQD CONSUHPTION P DECREASED UATER CONSUM?TfC?J P SCNEDULED Ei,KUAHASEk P

---------c-------c_--------------------------------- _________^^__1L_____--------------------------------------------------------- GME CODE: l-S&l-Gffl 2-f@&g?&jj~ 3-$Iya P-pgESzbz

SLI STUDY NO.: 3044;697 CLIENT: HCJNSANTO COHPANY


(POSITIVE FINDIKS)

PAGE 15

-------_--__---__--I________I___________-~------------------------- ----c---------c--c--------- _--_-__-^I___-------_____________I_

ANIMAt CLINICAL DAY OF 233333333334444444444 NO. OBSERVATIONS STUDY 901234567890123456789

----I----__----cI-________I____________^-~------------------------------------------------------------------- __L_-c-I^-_-----..c--

4618 F GROUP 4: 5713C-H SCHEDULEDEUTXANASIA P

4621 F GROUP 4: 5713C-H FEW FECES FECES SMALL IN SIZE SCHEDULED EUTHANASIA

4622 F GROUE' 4: 5713C-II SOFT sTOOLs SCHEDULEDE~SIA

4625 F GROUP 4: 5713C-H FECAL STAIN SCHEDULEDEUTHANASIA

4636 F GROUP 4: 5713C-H SCHEDULED EUWANASIA

4638 F GROUP 4: 5713C-H URINE STAIN FECAL STAIN SCHEDULEDEIJTWWIA

P P

PP

P

P

P t p

P

'P .

4644 F GROUP 4: 5713C-H FEW FECES P DECREASED FOOD CONSUHPTION P SCHEDULEDEUTHANASIA P

I____-___LI__________I_____^^___________----------------------------------------------------------------------------------------- GRADE CODE: l-SLIGHT 2-HODERATE 3-HARKED P-PRESENT

t

SLI Study No. 3044.697 i : .* (92)

APPENDIX F

Individual 8ody Weight Data

. . ___.

SLI S'IUDY NO.: 3044.697 A PASSIVE CUTAHEiOUS ANAPHYLBXS ASSAY IN GUINEA PIGS CLIENT: liONSANT COHPANY INDIVIDUAL BODY WEIGHT DATA (GRAHS)

PAGE 1

-----___-________________^______________---------------------------------------~------------------------------------------- -e-e.--

DAY * 0 7 14 17 21 24 28 35 38 42 49 --I_cc---__----el---_I------------I----------------------------------------------------------- --------------------------- -----...------ GROUP 1: 5713C-I (lo%)

4606 F 315 352 391 389 400 421 442 372 399 469 453 4610 F 335 378 400 411 423 431 458 411 465 487 504 4619 F 333 374 396 393 419 435 451 455 477 501 524 a. 4626 F 322 361 381 389 416 427 439 463 484 512 527 : 4627 F 319 370 408 418 436 .457 468 435 504 522 518 4633 F 327 376 397 399 431 426 452 426 427 455 474 4635 F 340 386 408 428 451 466 484 488 492 509 537 4642 F 345 408 434 462 482 485 516 453 491 527 548 4643 F 345 395 428 455 483 .494 508 525 526 554 607 4649 F 340 388 435 442 486 478 507 503 523 547 598

WAN 332 379 408 419 443 452 473 453 479 508 529 E S.D. 10.8 16.3 18.7 27.3 31.2 27.4 29.1 45.2 40.0 31.7 48.1

N 10 10 .lO 10 10 10 10 10 10 LO 10 ------------------^-I__c__________c_____-----------------------------~----------------------- _________I---------c----------------

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAFHYLAXIS ASSAY IN GUINEA PIGS CLIENT: HONSAKMCOHPANY INDIVIDUAL BODY VEIGHT DATA (GRAMS)

PAGE 2

---------------------------------------------------------------------------------------------------------------------------------

(FOUNDDEAD) DAY 0 7 14 17 21 24 27 28 35 38 42 49

i' ------------------_________ c-------------------________I___________-------------------------------------------------------------- GROUP 2: 5713C-K (10%)

4602 F 4608 F 4609 F 4611 F 4617 F 4620 F 4632 F 4639 F 4641 F 4645 F

344 392 403 401 399 429 - 461 448 497 515 518 334 342 348 346 340 363 - 400 393 401 400 424 320 334 347 332 354 385 - 400 411 448 476 471 326 338 350 343 352 326 279 - - - - - 343 330 330 336 352 381 - 421 448 444 453 434 313 316 320 323 320 318 - 356 306 315 351 372 336 334 338 334 344 354 - 369 379 375 400 390 340 333 347 350 364 384 - 404 355 345 362 389 328 336 312 332 341 382 - 403 .434 445 453 466 347 362 364 361 380 410 - 434 451 470 496 519

IHUN 333 342 346 346 355 '373 - 405 403 416 434 443 S.D. 11.1 21.0 25.3 22.2 22.2 34.3 31.6 49.7 60.5 58.4 54.7

N 10 10 10 10 10 10 9 9 9 9 9 --------------------_____^________^_____----------------------------------------------------------------------------------------- NOTE: THE XEAN IS NOT CALCULATED FOR ANIHALS FOUND DEAD.


A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINF.A PIGS INDIVIDUAL BODY WEIGHT DATA (GRAHS)

. PAGE 3

----------------________________________-----------------------------*----------------------------------------------------------- DAY 0 7 14 17 21 24 28 35 38 42 49

----------------------------^------------------------------~---------------------------------------------------------------- _-m---- . . GROUP 3: 5713~~H (10%)

4612 F 326 336 366 359 370 395 4623 F 319 334 360 343 363 378 4628 F 316 324 340 332 352 377 4629 F 340 335 367 340 363 386 4630 F 334 338 351 340 349 385 4640 F 343 351 367 358 373 406 4646 F 332 339 353 332 352 369 4647 F 342 352 349 356 382 418 4648 F 347 348 367 358 340 322 4650 F 335 332 338 336 344 371

416 399 393 417 412 431 389 441 321b 402

424 453 421 436 403 410 412 447 413 403 419 480 398 436 450 481

355 384

459 487 452 470 430 454 465 470 421 426 515a 517 463 465 470 494

429 411

HEAN 333 339 356 345 359 381 402 411 437 456 466 S.D. 10.4 8.9 11.3 11.2 13.4 25.8 32.8 25.6 33.2 28.4 32.8

N 10 10 10 , 10 10 10 10 9 9 9 9 --------------------------------------------------------------------------------------------------------------------------------- aADDITIONAL WEIGHT TAKEN ON DAY 43 (501 G) m ENSURE WEIGHT COLLECTED ON DAY 42 WAS NOT ABBERANT. %iIHAL WAS REMOVED FROM STUDY ON DA: 28, BUT WAS NOT EUTHANIZED UNTIL DAY 30 AT WHICH TME THE BODY WEIGHT WAS 345 G.

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS CLIENT: nmsm COHPANY INDIVIDUAL E!ODY WEIGHT DATA (GRAM)

PAGE 4

---------------------------------------------- __-________---cI________________c_______-------------------------------------------

DAY 0 7 14 17 21 24 28 35 38 42 49 --------------------------------------------------------------------------------------------------------------------------------- GROUP 4: 5713C-H

4601 F 326 355 406 405 435 452 469 496 502 521 526 4604 F 344 372 420 420 454 463 490 428 500 552 563 4614 F 328 351 381 397 412 435 445 480 498 529 499 4618 F 343 375 428 441 463 489 524 548 573 590 596 4621 F 339 389 434 452 476 493 496 543 557 573 586 4622 I? 318 353 379 392 413 417 440 456 483 489 499 4625 F 347 389 439 465 464 507 545 575 582 610 628 4636 F 334 373 412 443 482 473 500 520 566 562 594 4638 F 334 366 410 414 443 462 489 509 536 544 550 4644 F 319 340 373 382 394 415 437 450 455 478 468

HEAN 333 366 408 421 444 461 484 501 525 545 551 S.D. 10.3 16.4 23.6 27.9 29.6 31.4 36.0 47.5 43.4 41.9 51.7

N 10 10 10 10 10 10 10 10 10 10 10 ---------------------------------------------------------------------------------------------------------------------------- -----


APPENDIX G

Individual Body Weight Gain Data

-,

-

-

-

-

-

-

-

-

-

-

SLI STUDY NO.: 3044.697 CLIENT: lmNsmcowANY

A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS INDIVIDUAL BODY WFJGHT GAIN DATA (GRAHS)

PAGE 1

-------------------____^L_______________-----------------------------------------------------------------------------------------

DAY o-7 7-14 14-17 17-21 21-24 24-28 28-35 35-38 38-42 42-49 i’ -----------------------------------------------------------------------------------------------------------,---------------------- GROUP 1: 5713C-I (10%)

4606 F 37 39 -2 11 21 21 -70 27 70 -16 4610 F 43 22 11 12 8 27 -47 54 22 17 4619 F 41 22 -3 26 16 16 4 22 24 23 4626 F 39 20 8 27 11 12 24 21 28 15 4627 F 51 38 10 18 21 11 -33 69 18 -4 4633 F 21

:“6 22 2 32 -5 26 -26 1 28 19

4635 F 20 23 15 18 4 4 17 28 4642 F 63 26 28 20 3 31’ -63 38 36 21 4643 F 50 33 27 28 11 14 17 1 28 53 4649 F 48 47 7 44 -8 29 -4 20 24 51

nhw 47 29 11 24 9 21 -19 26 30 21 z+ S.D. 7.5 9.6 11.0 9.8 10.0 7.4 33.3 22.6 15.3 21.2

N 10 10 10 10 10 10 10 10 10 10 ---------------------------------------------------------------------------------------------------------------------------------

SLI SWDY NO. : 3044-697 A PASSIVE CUTANEOUS ANAPHiLAXIS ASSAY IN GUINEA PIGS CLIENT: HONSANTO COHPANY INDIVIDUAL BODY WEIGiiT GAIN DATA (GRAMS)

PAGE 2

--------------------______I____I________----------------------------------------------------------- -_____-------,-,--------------

DAY o-7 7-14 14-17 17-21 21-24 24-28 28-35 35-38 38-42 42-49 --------------------------------------------------------------------------------------------------------------------------------- GROUP 2: 5713C-K (10%)

4602 F 48 11 -2 -2 30 4608 F a 6 -2 -6 23 4609 F 14 13 -15 22 31 4611 F 12 12 -7 9 -26 4617 F -13 - 0 6 16 29 4620 F 3 4 3 -3 -2 4632 F -2 4 -4 10 10 4639 F -7 14 3 14 20 4641 F a -24 20 9 41 4645 F 15 2 -3 19 30

32 -13 49 18 3 37 -7 8 -1 24 15 11 37 28 -5

40 38 15

2 24

27 -4 9 -19 -50 9 36 21 10 -4 25 -10

-49 - .lO 17 27 31 11 a 13 17 19 26 23

HEAN 9 4 0 9 19 27 -3 13 18 9 S.D. 16.6 11.1 9.2 9.6 19.8 10.0 30.1 19.6 11.6 16.9

N 10 10 10 10 10 9 9 9 9 9 ---------------------------------------------------------------------------------------------------------------------------------

I

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS PAGE 3 CLIExk IK?NsmcoKPANY INDIVIDUAL BODY WEIGHT GAT'N DATA (GRAHS)

-------------------------------------------------------------------------“------------------------------------------------- ------

DAY o-7 7-14 14-17 17-21 21-24 24-28 28-35 35-38 38-42 42-49 --------------------_____I______________---------------------------------------------------------- _______L____----_---__I________ GROUP 3: 5713C-H (10%)

4612 F 10 30 -7 11 25 21 4623 F 15 26 -17 20 15 21 4628 F 8 16 -8 20 25 16. 4629 F -5 32 -27 23 23 31 4630 F 4 13 -11 9 36 27 4640 F 8 16 -9 15 33 25 4646 F 7 14 -21 20 17 20 4647 F 10 -3 7 26 36 23 4648 F 1 19 -9 -18 - 48 -1 4650 F -3 6 -2 a 27 31

8 29 6 22 15 16 10 7 20 -5 35 ia 1 -10 ia

-12 61 35 9 38 27 9 31 -11

-47 29 45 -

28 18 24

: 2 2

24

da

HEXN 6 17 -10 13 22 21 a -1 26 19 10 S-D. 6.2 10.7 9.6 12.6 15.7 9.2 20.0 20.2 16.1 14.8

N 10 10 10 10 10 10 9 9 9 9 ----------_--I------__________________c_----------------------------------------------------------------------------------- ------

SLI STUDY NO.: 3044.697 A PASSIVE CUTANECIUS ANM'HYLAXIS ASSAY IN GUINEA PIGS CLIENT: )IoNsm COKPANY RiDIvIDUAL BODY WEIGHT GAIN DATA (GRAHS)

PAGE 4.

----------------I--------------------------~--------------------------------------------------------- _-__----- -‘_------ ---- -e-e-.--

DAY o-7 7-14 14-17 17-21 21-24 24-20 28-35 35-38 38-42 42-49 ----------c---^---_-___________^_^______------------------------------------------------ ________________----___^________________- GROUP 4: 5713C-H

4601 F 4604 F 4614 F 4618 F 4621 F 4622 F 4625 F 4636 F 4638 F 4644 F

29 51 -1 30 17 17 27 6 19 5 28 48 0 34 9 27 -62 72 52 11 23 30 16 15 23 10 35 18 31 -30. 32 53 13 22 26 35 24 25 17 6 50 45 18 24 17 3 47 14 16 13 35 26 13 21 4 23 16 27 6 10 42 50 26 -1 '43 38 30 7 28 18 39 39 31 39 -9 27 20 46 -4 32 32 44 4 29 19 27 20 27 8 6 21 33 9 12 21 22 13 5 23 -10

HEAN 33 42 13 23 17 23 17 25 20 6 S.D. 8.8 9.5 10.5 11.7 13.8 10.7 29.5 20.8 15.5 16.5

N 10 10 10 10 10 10 10 10 10 10 ----------------_-__------------------------------------------------------------------------------ ________------------________c__

c SU Study No. 3044.697 (~02)

APPENDIX ii

Individual Hematology Data

;:, ,: i

::a

^_... - ._..__ .- -

:-_: &p

-- .- ._

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GllINEti PIGS CLIENT: HONSANTO COHPANY INDIVIDUALHEHATOLOCYDATA

PAGE 3

DAY 21 1, -----------^-----^--______^I____________----------~------------------------------------------------------------------------------

HEAN HEAN HEAN SEGD ERYTHRO- HEMO- HEHA- CORPUS CORPUS CORPUS PLATE- NEUTRO- LYHPHO- HONO- BASO- EOSINO-

ANIHAL CYTES GIDBIN TOCXIT VOLUHE EMGLEN HEHO-CON LETS PHILS CYTES CYTES PHILS PHILS NO. 10*6/CHH G/DL % FL PC G/DL 10*3/CHH 'x WEK x wac x d3c x WBC x mc

--------------------_______c_____c______----------------------------------------------------------------------------------------- cxuP 2: 5713C-K (10%)

4602 F 4.80 12.6 39.2 81.6 26.2 32.2 350 4608 F 5.00 13.2 40.0 79.9 26.4 33.0 310 4609 F 4.80 13.5 41.2 85.8 28.1 32.8 640 4621 F 5.30 13.9 41.2 77.8 26.2 33.7 540 4617 F 4.90 13.1 40.3 82.2 26.7 32.5 320 4620 F 4.90 13.1 40.3 82.3 26.7 32.5 580 4632 F 5.80 15.0 46.1 79.5 25.9 32.5 540 4639 F 5.20 13.9 42.5 01-7 26.7 32.7 540 4641 F 4.80 13.2 39.4 82.1 27.5 33.5 580 4645 F 4.80 12.8 39.1 81.5 26.7 32.7 530

54 15 42 67 72 61

5": 56 82

44 2 83 1 57 1 30 3 26 2 36 3 57 2 43 0 43 1 17 1

HEAN 5.03 13.4 40.9 81.4 26.7 32.8 493 55 44 0 0 SJI. 0.323 0.69 2.10 2.11 0.65 0.47 119.5 18.8 18.8. 1.; 0.0 0.3 N 10 10 10 10 10 10 10' 10 10 10 10 10

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS CLIEiNT: HrnSANM COtIPANY INDIVIDUAL HEHATOIBGYDATA

PAGE 4

DAY 21 ;' 1-------------------___________________^--------------------------------------------------------------------------------- _--m-v--

NON-SEGD NUCLE- NON-SECD NEUTRO- ATED RETICU- LEUKO-

ANIMAL PHIIS R8C'S LOCYTES CYTEX No. XWBC % wJ3c x RBC 10*3/cm

---------------L----_____C______________---------------------------------------------------------------------------------- -em---- GROUP 2: 5713C-K (10%)

4602 F 4608 F 4609 F 4611 F 4617 F 4620 F 4632 F 4639 F 4641 F 4645 F

HEhN 0 0 1.6 12.00 S.D. 0.0 0.0 0.74 5.043 N 10 10 10 10

0 0.6 10.90 0 1.7 5.10 0 3.2 10.60 0 1.6 11.40 0 1.5 23.70 0 0.7 14.50 0 1.1 9.20 0 1.3 12.30 0 1.9 7.60 0 1.9 14.70

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS CLIENT: HONSANMCOHPANY INDIVIDUAL HEHATOLOGYDATA

PAGE 5

DAY 21 --------------_-___-______^__^__________-----------------------------------------------------------------------------------------

HEAN HEAN HEAN SEGD EEtYTHRO- HMO- HE?& CORPUS CORPUS CORPUS PLATE- NEUTRO- LYHPHO- HONO- BASO- FQSINO-

ANIUAL CYT.ES GLOBIN MCRIT VOLUHE HHGLBN HEHO- CON LETS PHILS CYTES CYTES PHILS PHILS No. 10*6/CHH G/DL x FL PG G/DL 10*3/cHH % WBC 1 WBC % WBC % WBC % WBC

---------c----------------------------------------------------------------------------------------------------------------------- GROUP 3: 5713C-H (10%)

4612 F 5.10 14.4 42.3 83.0 28.2 34.0 4623 F 4.70 12.7 37.6 79.9 27.0 33.8 4628 F 4.20 11.6 34.5 82.2 27.6 33.6

' 4629 F 4.40 11.7 35.2 80.1 26.6 33.2 4630 F 5.10 14.0 42.4 83.2 27.5 33.0 4640 F 4.80 12.8 39.1 al.4 26.7 32.-a 4646 F 4.80 13.2 39.2 81.7 27.5 33.7 4647 F 4.70 12.9 39.2 83.4 27.4 32.9 4648'3 5.20 14.0 42.8 82.3 26.9 32.7 4650 F 4.70 12.4 37.7 80.3 26.4 32.9

350 29 69 1 0 590 42 53 4 0 380 68 28 3 0 430 68 30 2 0 490 68 31 1 0 410 69 29 2 0 600 64 33 1 0 550 93 7 0 0 510 57 41 2 0 390 62 37 1 0

nm 4.77 13.0 39.0 81.8 27.2 33.3 470 62 36 2 0 1 S.D. 0.313 0.95 2.89 1.30 0.55 0.47 90.6 17.1 16.4 1.2 0.0 0.7 N 10 10 10 10 10 10 10 10 10 10 10 10

I I

SLI S’lUDY NO. : 3044.697 .A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS CLIENT: HoNsANTocoNpANY INDIVIDUALHEXA'i'OUX;YDATA

PAGE 6

DAY 21 --------------------1___________________-------------------------------------------------------------------------------~---------

NON-SEGD NUCLE- NON-SEGD NEUTRO- ATED RETICU- LEUKO-

ANIHAL PHIIS RBC'S I.4lcYTEs CYTES NO. x WBC % WBC % RBC 10*3/m

----------------^---_________________^I_ ----------_--------^-~------------------------------------------~------------------------ GROUP 3: 5713c-H (10%)

4612 F 4623 F 4628 F 4629 F ,463O F 4640 F 4646 F 4647 F 4648 F 4650 F

0 0 0 0 0 0 0 0 0 0

0.9 1.9 0.6 0.8 1.7 1.2 0.8 1.2

9.40 6.80

19.40 20.50 10.00 12.90 13.40 26.90 10.10 12.80

HE4N 0 0 1.2 14.22 S-D. 0.0 0.0 0.58 6.187 N 10 10 10 10

--------------------_________________l_l-----------------------------------------------------------------------------------------

--- -- - _- -._._ _ .~---- - _-“. - _-.-- _ _

SLI STlJDY MO.: 3044.697 A PASSIVE CUTANEOUS ANAPHYIdXIS ASSAY IN GUINEA PIGS CLIENT: HONSANTO COKPANY INDIWDUALHEHA'NXOGYDATA

PAGE 7

DAY 21 ---------_------___________________c____-----------------------------------------------------------------------------------------

HEAN frEAN HEAN SEGD ERYTHRO- HMO- HEHA- CORPUS CORPUS CORPUS PLATE- NEOTRO- LYHE'HO- HONO- BAso- mstio-

ANIHAL CYT.ES GLOBIN MCRIT VOLUHE HHGLBN HEHO-CON LETS PHILS CYTES CYTES PHILS PHILS NO. 10*6/CHH G/DL X FL PG G/DL 10*3/C&f % WBC % WBC % w8c % WBC % WBC

---------_----__________________I_______----------------------------------------------------------------------------------------- GROUP 4: 5713C-H

4601 F 5.40 15.2 4604 F 4.80 13.3 4614 F 5.10 13.7 4618 F 5.00 15.1 4621 F 4.90 13.9 4622 F 5.10 14.2 4625 F 4.90 14.2 4636 F 4.90 13.9 4638 F 4.90 13.8 4444 F 5.50 15.2

NEAN 5.05 14.3 S.D, 0.232 0.68 N 10 20

46.1 85.4 28.1 33.0, 600 1 0 1 41.1 85.6 27.7 32.4 690 0 0 1 41.5 81.3 26.9 33.0 630 39 61 0 0 45.0 90.1 30.2 33.5 540 41 56 x 1 2 42.5 86.7 28.4 32.7 250 23 74 2 0 1 42.3 83.0 27.8 33.5 610 41 56 0 0 3 42.5 86.7 29.0 33.4 530 44 56 0 0 0 42.1 86.0 28.4 33.0 590 30 68 1 0 1 41.7 85.1 28.2 33.1 510 21 76 0 0 3 45.9 83.5 27.6 33.1 610 17 78 1 0 4

43.1 85.3 28.2 33.1 556 30 1.87 2.41 0.89 0.35 119.7 10.4

97 0.: 0.: 2 1.3 c

10 10 10 10 10 10 10‘ 10 10 10

- 2

k2 -J

I 1

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAPHYLMIS ASSAY IN GUINEA PIGS CLIENT: HwwTO COHPANY INDIVIDUAL HEWTOiOGY DATA

PAGE 8

DAY 21 --------------------------------------------------------------------------------------------------------------------------- -----..

NON-SEGD NUCLE- NON-SEGD NEUTRO- ATED RETICU- LEUKO-

ANIHAL PHILS RBC'S LOCYTES CYTES NO. x WBC x WBC x RBC 10*3/cHH

--------------------------------------------------------------------------------------------------------------------------- e--e-- GROUP 4: 5713C-H

4601 F 4604 F 4614 F 4618 F 4621 F 4622 F 4625 F 4636 F 4638 F 4644 F

1.1 4.60 1.4 7.10 2.0 6.20 3.2 8.10 2.5 5.90 1.0 7.00 1.9 6.70 1.5 5.30 1.1 6.80 1.9 6.70

HEAN 0.0" 0.0" 1.8 6.44 S-D. 0.70 0.989 N 10 10 10 10

--------------------__I_________________-------------- ---------,,------,-‘--,---------,--,----------------------------------------------

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS CLIENT: HONSANM COHPANY INDIVIDUAL HEXATOuK;Y DATA

PAGE 1

DAY 21 --------------------_^___ c-------------------_______________I____----------------------------------------------------------------

nJ3AN HEAN HEhN SEGD ERYTHRO- HMO- HEHA- COEWS CORPUS CORPUS PLATE- NEIJTRO- LYHE'HO- HONO- BASO- EXMNO-

ANIHAL CYTES- GLI)BIN TOCRIT VOLUHE HHGLW HEHO4JON LETS PHILS CYTES CYTES PHILS PHILS No. 10*6/C?iH G/DL % FL PG G/DL 10*3/m % WBC x WBC x WBC x WBC x WBC

--------------------______ -----_------------------------------------------------------------------------------------------------- STOCK

4603 F 5.00 14.1 42.5 85.1 28.2 33.1 430 28 JO 1 0 1 4605 F 5.10 14.7 43.3 .85.0 28.8 33.9 540 29 68 0 0 3 4607 F 4.80 14.1 42.4 88.4 29.4 33.2 390 21 78 1 0 0 4613 F 5.50 15.0 45.5 82.7 27.3 33.0 510 36 62 1 0 1 4615 F 5.40 14.7 44.8 82.9 27.2 32.8 540 31 67 0 0 2 HEAN 5.'16 14.5 43.7 84.8 28.2 33.2 482 29 69 1 0 1 - .A. S.D. 0.288 0.40 l-39 2.30 0.95 0.42 68.3 5.4 5.8 0.5 0.0 1.1 .24 N 5 5 5 5

5 5 5 5 5 .5 5 5 V 2

I -I I I I

SLI Study No. 3044.697 (113)

-

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-

APPENDIX I

Individual RBC Morphology Data

F F

i.

I

i I

i I

i I I I I

i E ’ b

I’,

. . ii?

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS 'CLIENT: HONSANTO COHPANY INDIVIDUAL RBC HORPHOL DATA

PAGE 2

DAY 21 ---c-- ---------------_------------------------------------------------------ -------- --__---_------------------ __c__-_-I-------.---

POLY- ANIHAL RBC HOR HACRO- HICRO- POIKILO- SPHERO- OVALO- HYPO- CHROH- NO. PHOLOGY CYTES CYTES CYlXS CYTES CYTES CHROHIC ASIA HISCEUANEOUS

__L___________I1________I___L___________----------------------------------------------------- ____^______c_____-__----------------

GROUP 2: 5713C-K (10%)

4602 F N 4608 F N 4609 F N 4611 F N 4617 F N 4620 F N 4632 F N 4639 F N 4641 F N 4645 F N

^_________________________________Ic -_--- --4--- _-^__-_________-________________________------------------------------------------

N = NORMAL 1+ = SLIGHT 2+ = HODEXATE 3t = HODERATE TO HARKED 4t = HARKED

.--

c

A PASSIVE CUTANEOUS ANAPHYMXIS ASSAY IN GUINEA PIGS INDIVIDUAL RBC HORPHOLOGY DATA

PAGE 3

DAY 21 i'

--------------------________C__I______L_--------------------------------------------------------- ____I__-_---I-c--_-------------- POLY-

ANIHAL RBC HOR HACRO- HICRO- POIKILO- SPHERO- OVAL& HYPO- WON- NO. PHOL&Y CYTES CYTES CYTES CYTES CYTES CHROHIC ASIA HISCELLANEOUS

-------------^-------------------------------------------------------------------------------------------------------------------

GROUP 3: 5713C-H (10%)

4612 F N 4623 F N 4628 F N 4629 F N 4630 F N 4640 F N 4646 F N 4647 F N 4648 F N 4650 F N

--------------------___c________________-----------------------*-------------------------------------- _-__-_---------------------

N = NORHAL li- = SLIGHT 2i- = HODERATE 3+ = MODERATE TO HANQ.D 4t=HARKED

SLI Study No. 3044.697 (119)

APPENDIX J

Individual Biochemistry Data

-

-

-

-

-

-

-

. .

SLI S’IUDY NO. :, 3044.697 CLIENT: HQNSANTO COHPANY

DhY 21

A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS INDIVIDUAL BIOCHMISTRY DATA

PAGE 1

---------------------------------------------------------------------------------------------------------------------------------

PO4 7xrI.u hLK PHOS UREA CHLO- CHOLE- CREA- POTAS- PHOS- BIiI- TOTAL

hNlXhL 'ThSE NITROGEN ChLCIuH RIDE STEROL TININE AST SIIJH SODIUH PHORUS RUBIN PROTEIN No. IWL HGIDL MG/DL tfHOL/L HG/DL NG/DL IWL HMOL/L Ht+OL/L HG/DL MG/DL G/DL

-----------------c--_________^__________-------*--------------------------------------------------------------------------------- GROUP 1: 5713C-I (10X)

4606 F 203 20 11.22 112 44 0.35 4610 F 156 17 10.62 109 43 0.37 4619 F 142 18 10.98 114 68 0.39 4626 F 185 18 10.84 115 53 0.34 4627 F 155 21 11.22 116 58 0.26 4633 F 159 20 9.96 111 48 0.36 4635 F 259 21 11.16 113 51 0.40 4642 F 189 18 ‘ 11.63 114 46 0.40 4643 F 185 21 11.98 115 48 0.49 4649 F 174 20 11.27 113 42 0.51

39 70 76 97 54 48 46

194

4.81 143 5.8 0.50 4.62 5.10 141 5.4 0.41 4.70 4.45 141 6.5 0.48 4.96 5.29 137 6.6 0.67 4.68 5.30 140 6.7 0.57 4.92 4.78 140 6.6 0.41 4.97 5.08 138 6.6 0.43 4.30 5.32 142 7.5 0.55 4.86 5.94 141 6.8 0.51 4.78 5.54 138 7.0 0.50 4.53

UEhN 181 19 11.09 113 50 0.39 72 5.16 140 6.6 0.50 4.73 S-D. 33.4 1.5 0.551 2.2 7.9 0.071 46.7 -0.423 1.8 0.59 0.080 0.212 N 10 10 10 10 10 10 10 10 10 10 10 10

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS hNAPBYLAXIS ASSAY IN GUINEA PIGS CLIENT: HONShNTo COMPANY mrnVrnu& BIOCHEHISTRY DATA

PAGE 2

DAY 21 --.-------------------------------------------------------------------------------------------------------------------------------

GU)B hNrHhL h/G GLUCOSE ULM ALT ALEWHIN

No. RATIO tlG/DL G/DL IWL G/DL ------.,--__---__ --------------------___________l_______l-------------------------------------------------------------------------

GROUP 1: 5713C-I (102)

4606 F 1.16 126 2.14 4610 F 0.97 122 2.39 4619 F 1.00 135 2.48 4626 F 1.03 172 2.31 4627 F 1.11 168 2.33 4633 F 1.11 147 2.35 4635 F 0.98 202 2.18 4642 F 1.13 138 2.28 4643 F 1.15 142 2.22 4649 F 1.16 137 2.10

28 2.48 27 2.31 36 2.48 39 2.37 34 2.59

3: 2.61 2.13

58 2.57 28 2.56 28 2.43

HEhN 1.08 149 2.28 34 2.45 S-D. 0.077 24.7 0.119 9.3 0.151 N 10 10 10 10 10

---------------------------------------------------------------------------------------------------------------------------------

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAFHYLAXIS ASSAY IN GUINEA PIGS PAGE 3 CLIENT: MGNShNTOCOBPhNY INDIVIDUAL BIOCIiEHISTRY DATA

DAY 21 . ---------------------------------------------------------------------------------------------------------------------------------

PO4 lY0Th.L hLK PHOS UREA CHLO- CHOLE- CREh- POThS- PHOS- BILI- XlThL

hNmhL .'ThSE NITROGEN CALCIUB RIDE STEROL TININE AST SIUB SODIDH PHORUS RUBIN PROTEIN No. IWL HG/DL HG/DL HBOL/L' tlG/DL BG/DL IWL BBOL/L BBOWL HG/DL HG/DL G/DL

--------------------__c__I______________----------------------------------------------------------------------------------------- GROUP 2: 5713C-K (10%)

4602 F 144 4608 F 84 4609 F 151 4611 F 131 4617 F 113 4620 F 103 4632 F 124 4639 F 142 4641 F 114 4645 F 123

21 17 19 21 18

ii 20 16 19

11.68 109 63 0.41 73 5.37 139 10.63 1 106 22 0.46 133 5.12 137 11.43 113 36 0.26 67 5.34 143 11.45 111 49 0.40 88 5.59 141 11.72 113 26 0.34 61 4.83 143 11.26 112 . ,28 0.67 100 4.76 142 10.85 115 45 0.41 59 4.75 144 10.69 110 42 ' 0.37 101 4.64 138 10.97 107 37 0.32 90 4.44 140 10.92 107 33 0.39 64 4.75 136

6.9 0.72 4.86 4.6 0.46 4.16 5.4 0.62 4.26 5.6 0.71 4.43

::; 0.50 4.57 0126 4.79

5.0 0.57 4.24 6.0 0.48 3.99 6.2 0.49 4.63 6.1 0.56 5.28

HEhN 123 S-D. 20.4 N 10

19 11.16 110 38 0.41 a4 4.96 140 5.7 0.54 4.52 2.0 0.401 3.1 12.3 0.110 23.4 0.373 2.8 0.64 0.135 0.385 10 10 10 10 10 10 10 10. LO 10 10

----------------1---_^__________________-----------------------------------------------------------------------------------------

SLI S’iUDY NO.: 3044.697 CLIENT: HONSANTO COHPANY A PASSIVE CUTANEOUS ANAl'HYLAlfIS ASSAY IN GUINEA PIGS

INDIVIDUAL BIOCHEHISTRY DATA PAGE 4

DAY 21 ------------__-__________ ----------_-______-___^____ --------c---_-_-_-__________________^ _^-c_____--__I_I^__---------------------

ANIHAL GtoB A/G

NO. GLUCOSE ULIN

RATIO tfG/DL ALT ALBiJHIN

G/DL IU/L . ..-----a- G/DL -------------______________ GROUP 2: 5713C-K (10X)

------------------____ ------_-_-_---___________________l_l____- __-----------------------------

4602 F 0.98 4608 F

145 2.46 1.18

4609 F 164 1.91

1.06 147 4611 F 2.07

1.14 4617 F

138 2.07 I.04

4620 F 145 2.24

l-10 4632 F

156 2.28 1.06 143

4639 F 2.05 1.12 131

4641 F 1.88 0.88 138

4645 F 2.46 0.88 157 2.81

83 2.41 138 2.26 46 2.19 50 2.36 41 2.32

137 2.51 36 2.18 46 2.11 53 2.16 53 2.47

nut4 1.04 146 S.D. 2.22 0.104 9.9

2.30 N 0.288

10

386:

10 0.138

10 10 10 --------

------_-_L_____^-________I

____-______-I___-^_-_____

______________-__-_-_______

___i-________-______^_______

---------------

- ;o” w

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAPBYLAXIS ASSAY IN GUINEA PIGS CLIENT: HONsANTOcoBFANY INDIVIDUAL BIOCHEBISTRY DATA

PAGE .5

DAY 21 -----------^------*-_I__________________------ __,-,,__---,_,__,,,,,,,,____,,,,,,,,,,,,---------------------------~---------------

PO4 MTkL ALK PHOS UREA CHm- CHOLE- CREA- POTAS- PHOS- BILI- !WJXL

ANIBAL 'TASE NITROGEN CALCIUB RIDE STEROL TINTNE PST SIUH SODIUH PHORUS RUBIN PRd'TEm NO. IU/L HG/DL HG/DL BHOL/L HG/DL HG/'DL W/L ttHOL/L HHOWL HG/DL HG/DL G/DL

--------------I---I-_____I_ --------------------_______________I____-------------------------------------------------------------- GROUP 3: 5713C-H (10%)

4612 F 123 '12 10.91 110 34 0.38 52 3.71 140 5.7 0.64 4.27 4623 F 102 12 10.91 107 29 0.33 55 4.89 140 5.6 0.57 4.06 4628 F 105 11 11.21 105 29 0.42 103 4.60 136 6.0 0.59 4.09 4629 F 136 11 10.24 106 29 0.26 84 4.81 135 5.8 0.64 3.69 4630 F 143 11 10.81 109 26 0.29 88 4.85 138 5.3 0.60 4.12 4640 F 142 8 10.39 109 35 0.44 68 4.64 138 5.2 0.49 4.28 4646 F 112 13 10.66 108 40 0.38 76 4.45 139 5.0 0.53 3.79 4647 F 183 11 10.37 105 29 0.50 114 5.33 -139 6.2 0.56 3.65 4648 F 81 18 11.02 111. 54 0.39 113 5.32 136 6.0 0.56 4.45 4650 F 114 14 10.67 111 35 0.38 139 4.49 138 4.4 0.44 3.98

HEAN 124 12 10.72 108 34 0.38 89 4.71 138 5.5 0.56 4.04 S.D. 28.4 2.6 0.313 2.2 8.1 0.072 28.1 0.466 1.8 0.55 0.063 0.263 N 10 10 10 10 10 10 10 10 10 10 10 10

SLI STUDY NO.: 3044.697, A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS CLIENT: HQNSANTO COHPANY INDIVIDUAL BIOCBEHISTRY DATA

PAGE 6

DAY 21 ------c------------------------------------------------------------------------------------------------------------------- -e-w---

GIDB ANIML A/G GLUCOSE ULIN ALT ALBUHIN

No. RATIO . tfG/DL G/DL IU/L G/DL ---------------------------------------------------------------------------------------------------------------------------------

GROUP 3: 5713C-N (10x)

4612 F 1.03 4623 F 1.18 4628 F 1.10 4629 F 1.11 4630 F ' 1.16 4640 F 0.96 4646 F 1.01 4647 F 1.03 4648 F 1.06 4650 F 1.12

126 2.10 37 2.16 137 1.86 42 2.20 171 1.95 72 2.14 154 1.75 50 1.94 130 1.91 72 2.21 164 2.19 52 2.09 139 1.88 i 43 1.90 146 1.80 52 1.85 144 2.16 55 2.29 157 1.88 88 2.10

HEIN 1.08 147 1.95 57 2.09 2 S.D. 0.071 14.7 0.150 16.2 0.145 N 10 10 10 , 10 10

---------------------------------------------------------------------------------------------------------------------------------

I

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN GUINEA PIGS CLIENT: HONSANTO COBPANY INDIVIDUAL BIOCBEBISTRY DATA

PAGE 7

DAY 21 ---------*----------________________I___-----------------------------------------------------------------------------------------

PO4 mA.L AU PHOS UREA cHu)- CHOLE- CREA- POTAS- PHOS- BILI- TOTAL

ANIBAL 'TASE NITROGEN CALCIUB RIDE STEROL TININE AST SIUH SODIUB PHORUS RUBIN PROTEIN No. IWL KG/DL HG/DL BHOL/L HG/DL BG/DL IU/L BBOL/L BBOWL HG/DL HG/DL G/DL

---------------_----_____I_ ------------------------------------------------------------------------------------------------------ GROUP 4: 5713C-H

4601 F 174 21 11.31 115 4604 F 145 20 10.72 115 4614 F 157 21 11.27 113 4618 F 156 21 11.51 114 4621 F 221 22 11.59 120 4622 F 228 20 11.18 113 4625 F 182 22 11.85 112 4636 F 220 20 11.50 112 4638 F 159 22 11.23 112 4644 F 160 20 11.69 112

59 50 36 54

3": 47 51 24 55

0.40 32 3.81 142 6.8 0.49 5.18 0.38 50 5.25 138 7.5 0.53 4.94 0.45 42 4.70' 140 6.3 0.37 4.95 0.42 51 5.23 142 7.2 0.48 5.34 0.38 52 5.37 143 7.9 0.40 4.78 0.37 91 5.78 140 6.8 0.45 4.75 0.38 41 4.96 141 5.3 0.52 4.73 0.32 45 6.06 141 8.0 0.62 4.82 0.44 50 4.56 143 7.4 0.54 4.40 0.40 86 5.40 142 7.4 0.25 5.07

HEAN 180 21 11.38 114 45 0.39 54 5.11 141 7.1 0.47 4.90 S.D. 31.3 0.9 0.318 2.5 11.1 0.038 19.2 0.643 1.6 0.80 0.10s 0.264 N 10 10 10 10 10 10 10 10 10 10 10 10

---------------------------------------------------------------------------------------------------------------------------------

SLI S'IUDY NO.: 3044.697 A PASSIVE~CUTANEOUS AN4PHYLAXIS ASSAY IN GUINEA PIGS CLIENT:. NQNSANTO COKPANY INDIVIDUAL BIOCHEHIST+RY DATA

PAGE 8

DAY 21 ------------------------,,,,__--___-,,,,-------------------"-----------------------------------------------~---------------------

GLI)B ANIHAL A/G GLUCOSE ULIN ALT ALBUKIN

No. RATIO tlG/DL G/DL IU/L G/DL --------------------__________c_________-----------------------------------------------------------------------------------------

dROUP 4: 5713C-H

4601 F 1.06 159 2.52 28 2.67 4604 F 0.97 157 2.51 29 2.43 4614 F 1.03 135 2.44 35 2.51 4618 F 0.92 137 2.77 28 2.56 4621 F 1.15 145 2.23 30 2.56 4622 F 1.17 129 2.19 40 2.56 4625 F 1.14 174 2.21 32 2.52 4636 F 0.94 155 2.49 31 2.34 4638 F 0.99 152 2.20 43 2.19 4644 F 1.10 150 2.41 33 2.66

HEM? 1.05 149 2.40 33 2.50 S.D. 0.090 13.2 0.190 5.1 0.146 N 10 10 10 10 10

--------------------__^________________ -L------_-_-C--_----_________C__________---------------------------------------------- ----

I I I 1 \ .- I

SLI STUDY NO.: 3044.697 CLIENT: NWSANTC COBPANY

DAY 21

A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN~GUINEA PIGS IbDIVIDUAL BIOCBEHISTRY DATA

PAGE 1

-----------------_--_________ ---------------,----------------------------------------------------------------~------------------- PO4 TOTAL

ALK PHOS UREA CHID- CHOLE- CREA- POTAS- PHOS- BILI- mTAL ANIHAL 'TASE NITROGEN CALCIUB RIDE STEROL TININE AST SIUH SODIUB PBORUS RUBIN PROTEIN

No. IWL BG/DL HG/DL HHOWL HG/DL HG/DL IU/L HtlOL/L BBOL/L HG/DL HG/DL G/DL -------------------_______________ ------------^----------------------------------------------------------------------------------

SMCX

4603 F 213 18 11.10 104 41 0.43 39 4.80 136 5.8 0.36 4.37 4605 F 173 19 11.67 112 50 0.51 42 5.11 141 6.4 0.48 4.92 4607 F 172 19 11.10 110 70 0.32 46 5.00 137 6.4 0.59 4.55 4613 F 212 22 11.40 115 60 0.30 76 4.44 140 6.0 0.39 4.99 4615 F 143 18 11.39 113 50 0.35 44 5.11 140 7.4 0.60 4.63

HEAN 183 19 11.33 111' 54 0.38 49 4.89 139 6.4 0.48 4.69 S.D. 29.8 1.6 0.240 4.2 11.1 0.087 15.1 0.283 2.2 0.62 0.111 0.259 N S 5 5 5 5 5 5 5 5 5 5 S

--------------------__I_________________-----------------------------------------------------------------------------------------

SLI STUDY NO.: 3044.697 CLIENT: HONSANM COHPANY A PASSIVE CUTANFsOIJS ANAPHYLAXIS ASSAY IN GUINEA PIGS

INDIVXDUAL BIOCHWISTRY DATA PAGE 2

DAY 21 ---------_-_-_________ -----m-e .--------------- e---e

GIl)B -----l------f--_-____^___l_l______l_ ------------- -----I-------- _---------------

ANIHAL A/G No.

GLUCOSE ULIN BAT10 HG/DL

ALT ALBUMN WDL IWL -----mm- WDL ------e..--

SMCK -------------_-_-__________ ----------------__________I______ -------------------^---- ------------- --------------

4603 F 1.22 201 4605 F 1.07 1.97 29 155

2.40 4607 F 2.38 1.13 26

148 2.54

4613 F 2.14 0.91 29 201

2.41 4615 F 2.61 1.08 31

139 2.38

2.23 29 2.40 HEAN 1.08 169 S.D. 2.27 0.112 29

29.9 2.43 N 0.243 S 1.8

5 0.065

5 5 5 ---__---_______-_________ i-G ------me --------------------_________________ ----------...-- -2 ---^-------- _---___-_-_-^____-----------------

I I I I J .- 1 I 1 ! I I I ! I I 1 I I I I

i ;1

F: t i J

53-I Study No. 3044.697 (130)

APPENDIX K ’

Individual Gross Necropsy Observations

SLI STUDY NO.: 3044.697 A PASSIVE CUTANEOUS ANAPBYLAXIS ASSAY Ii GUINEA PIGS CLIENT: nmsmcoBPANY INDIVIDUAL GROSS NECROPSY OBSERVATIONS

PAGE 1

GRADE --------------------___I________________------------------------------------------------------------------------------------ --I-- ANIBAL NO. 4611 GROUP: 2 FEUALE FOUNDDEAD

WHOLE BODY GROSS: BODY FAT DEPLETION P LUNGS GROSS: DARK RED P

RIGHT APICAL LOBE STOtIACH GROSS: DISTE%DED P

WITH GAS AND GREEN FLUID

ANIHAL NO. 4648 GROUP: 3 FEHALE ABDOHINALCAVITY

TRACHEA

LUNGS

WHOLE BODY STOMACH

SHALL INTESTINE

EUTHANIZED HORIBUND GROSS: FLUID CONTENTS P

APPROXIHATELY 2.0 HL OF SLIGBTLY CLOUDY CLEAR FLUID GROSS: CONTENT ABNDRHAL P

ENTIRE LENGTH, WHITE FOAB GROSS: BOTTLED P

ALL U)BES, DARKREDAND RED GROSS: BODY FAT DEPLETION P 2 GROSS: DISTEt?DED P G, A

WITH GAS AND SMALL AMOUNT OF INGESTA V GROSS: DISTENDED P

ENTIRE TRACT, WITH GAS AND WATERY DIGESTA

GROSS GRADE CODE: l-SLIGHT, 2-NODERATE, 3-HARKED, P-PRESENT

I I ! I .I I I I I I 1 I 1 I 1 I I 1 I

SLI Study No. 3044.697 .(1.32)

Added Stock Animals

SLI STUDY NO.: 3044.697 CLIENT: HONSANTG COHPANY

A PASSIVE CUTANEOUS ANAPHYLAXIS ASSAY IN CUIt'=A PIGS INDIVIDUAL CLINICAL OBSERVATIONS

(POSITIVE FRQINGS)

PAGE 1

le___C_--..----- _____________--.c--- -_--..--c -------.---.-..-

__I_-c____I_________---------- ______Ic-____c______------------ -d-“-e ---s.-.w-

ANIHAL CLINICAL DAY OF 333334444444444 i'

NO. OBSERVATIONS STUDY 567890123456789 ."----c- __-_4---- _--_-m ____ -_-- _-_..e.-L------- __-______c____I_____________^__cc -m--m --me -_------ __fC-_-_---------

-----1--.----------

4603 F GROUP 5: 5713C-I (10%) FEW FECES PP

P SCHEDULED EiJ’IWWSJA

4605 F

4607 F

4615 F

4616 F

4624 F

4631 F

4634 F

GROUP 5: 5723C-I (IOr) FEU FECES FECES SHALL IN SIZE SCHEDULED EUTHANASIA

GROUP 5: 5713C-I (10%) SOFT STOOLS SCHEDULED EUTHANASIA

GROUP 5: 5713C-I (10X) FEfJ FECES SCHEDULED EUTHANASIA

GROUP 6: 5713C-H SCHEDULED EUTHANASIA

GROUP 6: 5713C-H SCHEDULED EUTHANASIA

GROUP 6: 5713C-H FEW FECES FECES SHALL IN SIZE SCHEDULEDEUTHANASIA

GROUP 6: 5713C-H FEW FECES ~-

PPP PPPP

P

P P

PP P

P

P

P PPPP

P

P P

SCHEDULED EUTHANASIA ______________-_-cII----- __c__________c__--c-__I__ -- _______^.e-----L-- --_--...-__-.-..--*-- -___________-___-I----- c---..-.-I-v--c.----.---

GRADE CODE: l-SLIGHT 2-HODERATE 3-HARKED P-PRESENT

_______-___------------- _--..-----------..---- --_-_____--_---_----_________ ---^----------_-----______^_____________---------------- 3 il ‘I tl N 6’ZE f’BE 8-x E’E’I *a-s 90s 069 S6f 805 rnH

E9P z!?J sstl ES!? d ST9tt ITS szs SIS 621; J LO9’1 LOS 68P SLP L6?7 d 5099 E’IS STS EES ESS d E09’1

(I.01) I-3&T/S :5 dnOXl ___-___^--...--.--- a-..--- ---."-.-------- __-__-__^__----_-c----------~---------- ------___---___-__-_-----------------------------------

6 L E 0 ma ------------- ______________^______I__c______c________---------- ------_--^^------^_-----------------------------------------------

‘I 33vd

kfxvm) vtwa m13h 4a0a wnaumm ANvdH~cNINvSNoH xftwi3 . s31d ~13~1113 M msv s~mdv~v snomvm wssvd v L69'11fOE : 'ON &ms 11s

------. __~~ ~--- ---_ - -__. --___- -.-.---.. .- -..--. .__

i

i i I Oi

I I

I I

i i f bYI I I I I I L

-

-

-

-

-

-

-

-

-

,

______I-____c___^__-______^__1__1_____1_-----------------------------------------------~ --------------------^_^___I__________^__-

, “,. +

.,,’ P N 8'CT E"lT 6’OT :a3 91: f- ST- Nvw

TZ ET- &!I ST98 ‘i- 0 tr - d L09+l 8T VT zz- d so9’1 82 8T- oz- d E098

(%OT) I-XTLS :5 dfl0'd3 ______________^____^----------------------------------------------------------------------------------- ----_--------------------- 6-f L-E E-O AVU _______________I_____l________l_________-------------~---------------------------------------------------------------------------

T XIV6 kxm3) vm tam mm ma wflamm

~3rd mm3 m mm sIxru.mw snommm uussvd $L ANvdHcn (llmv!TNoR w431?3 f 69 "1?0& :-ON UffLTnS 175

-

-.

-

-

-

-

7

(LE I->

SLI Study No. 3044.697 (139)

SLl PERSONNEL RESPONSIBILITIES -

Kimberly L. Bonnette, M.S., LATG

George A. Douds, MS.

Robert C. Springborn, Ph.D.

Malcolm Blair, Ph.D.

Joseph C. Siglin, Ph.D., DABT

Deborah A. Douds, M.S.

Pamela S. Smith, ALAT

Delores P. Knippen

Steven t-l. Magness, B.S., I-ATG

Jan K. Sever-t, B.S., ALAT

Anita M. Bosau, RQAP-GLP

Deanna M. Talerico, RQAP-GLP

Christopher W. Wilson, B.S., RQAP- GLP

J. Dale Thurman, D.V.M., M.S., DACVP .

Study Director/Manager of Acute Toxicology

Alternate Contact/Assistant Toxicologist

Chairman, President and CEO

Vice President and Managing Director

Director of Toxicoiogy

Assistant Manager of Acute Toxicology

Primary TechniciakSupervisor of Acute Toxicology

Supervisor of Pharmacy

Supervisor of Gross and Fetal Pathology

Supervisor of Acute Report Preparation

Director of Compliance Assurance

Supervisor of Nonacute Quality Assurance

Supervisor of Acute Quality Assurance

Director of Pathology


Appendix 7.3 Cottonseed Meal Analysis

Ralston Purina Ccg Checkerboard ST r Saint Louis. MO i

TO R-GOODMAN, .,, CC K. S. PHILLIPSI 2RS 0sG.HAUGHT-2E

-

0 LAB NO 604278 ENTERED 05/11/98 REPORTED 05114198

PO r ‘CSA’ NBP 6352322 COTTONSEED MEAL MONSANTO BBSG

-

LOW-LIMIT Z HIGH-LIMIT - --------- ---------o

10,oo

ASSAY -we--

MOISTURE AT ASSAY MOB

PROT.COMB.(N X 6.25) PRCB

AMMON I A AMIN

FAT (ETHER EXT.1 FTEE

FI8ER (CRUDE) FIBR

ASH ASHF

GOSSYPOL t FREE) GOSF

ANALY SI S --we----

8-36

23.0 % 20-O

a-i40 % 1 -aa -

13.8 % LO.00 20,o

19.4 % 20-a -/

5,oa 4.28 %

1.00 -

2-m 0,480 %

- THE LETTER CODE LOCATED BELOW EACH ASSAY IS A MFHOD REFERENCE CQDE- FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES l-800-423-6832

-,

Ralston Purina Cotnpany Checketioard Square Saint Louis, MCI 63164

TO R-GOODMAN,

I

CC Km S. PHILLIPS, ZRS D .G.HAUGHT-PE

F-3 s’ I

;LAB NO 6’06281 ENTERED'05/11/98 REPORTED 05/14/98

PO I ‘CS-D’ NB 6332332 CO7TONSEED MEAL MONSANTO BB5G

ASSAY CI,

c /-w--w

< !MOISTURE AT ASSAY MOB

PKOT.COMR.(N X 6,251

AMMN FAT (ETHER EXT,)

FTEE FIBER (CRUDE)

FIBR

ASHF GOSSYPOL (FREE)

GOSF c d

ANALY SI S UNITS LOU-LIMIT 2 HIGH-LIMIT ..-e"--w- ----- w..w---e.-- ---.m----w-

8.76 ‘% ~0.00

23.6

0,140

14-3

L8.0

4.21

0.400

%

%

%

%

%

r,

0

20.0

1.00

lO.00 20.0

20.0

5.00

l-00 . * 2.00

- THE LETTER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE, k’ FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES 1-800-423-6832 c I

-be lerm ‘Less Tnan” IS use0 IO SrgRlfy the lower llrrlt or c$Inr!tallon 01 lne Orwwufe unfjer me COnalllons emcxcvm. The use 01 the term ‘-Less Than” aoes nor lrmlV lnat

i XaCtIS at anawe we18 @resent. SsmDk~ Su’Jmwa 50 Rals:on Analvnczu ~aoora~w~e.s 10r tomme analySlS WI w retSinea tor a mIntmum ot ~n~ny 130) days aner VW recon ot ts issuQQ. Extended storage requirements must oa orouqfv to me ane*,lofl of Ralston AI-I~~AI~ I;lbora;orfes ~nor 70 or at the tlme ot sample sucm1s61on.

-

- Ralston Purina Compaf Checkerfioard Square Saint Louis, MO 63 364 -- -,a.

TO R -GOODMAN. CC K- S- PHILLIPS, 2RS DeG.HAUGHT-ZE

LAB NO, 604279 ENTERED OS/Ll/YS REPORTED 05/34/98

PO , “x5-B’ 6312323 COTTONSEED MEAL MONSANTO BB5G

ASSAY -a---


PROT-COMB. (N X 6,251 PRCB

AMMONI A AMMN

FAT (ETHER En-1 FTEE

FIBER (CRUDE) FIBR

ASH ASHF

GOSSYPOL (FREE) GOSi=

ANALY SI S UNITS -------- --em-

7.73 %

23.1

O-130

14-5

19-5

3-92

a.520

% 10,cJo

% 20-O

%

%

0

LOW-LIMIT 2 HXGH-LIMIT a--*---..w .w.m--------

LO.00

ta.0

1.00

20.0

5.00

2-00

THE LE-RER CODE LOCATED 8ELOW. EACH ASSAY IS A METHOD REFERENCE CODE. FOR ADDITIONAL INFORMATION CONTACT CL1 ENT SERVICES L-800-433-6832

-

-

-

-

-

-

-8

F


Monsanto Company MSL-15763 Biotech Regulatory Sciences Page #: 172 of 197

7.4 Feed Analysis Pre-study

Ralston fgfc$t& .

-

Ralston Purina Company _ Checkerboard Square Saint iauis, MO 63764

TO R-E-GOODMAN, cc K, S. PHILLIPS, 2RS

. . LAB NO 614489 ENTERED 07/l 5f 38 REPORTED 07/20/98

5713 I USDr’GUfNEA PIG DIET, ROOM TEMP. GROUP 4 MONSANTO 3f35G jmcw Bt ia%cz;~x -

GF/ ASSAY ANALYSIS UNITS LOW-LIMIT Z HIGH-LIMIT - --I-- ------s.- --B-w -w----m-- ----..----..

MO1 STURE AT ASSAY 8.00 % MOB

PROT.COMB.(N X 6.25) 20.6 % - PRCB

FAT (ETHER EXT,) 5.25 % FTEE -

FIBER (CRUDE) 16.2 % FIBR

ASH 7-73 % ASHF

I RON 298, PPM FEF

TOTAL VITAMIN C 1200. PPM VTCA

GOSSYPOL (FREE) 0.(3340 x 0,100 GOSF

I,

,r.

-

THE LEITER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE. FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES l-800-423-6832

F . + th,.:

: ,

Ralston Amilyticd Laboratories Ralston Purina Compwj

Checkerboard Square Saint Louis. MO 63164

TO R.E.GOODMANr CZ K, S. PHILLIPS. ZRS

LAB NO 616488 ENTE~ED”b7/15/98 REPORTED 07120198

5713H USD, GUINEA PIG DIET, ROOM TEMP. GROUP LO MONSANTO BB 5G

c&a b:& 8% 1

ASSAY eve-..



FAT ( ETHER EXT. 1 FTEE

FIBER (CRUDE) FSBR

ASH ASHF

I RON FEF

TOTAL VITAMIN C VTCA

GOSSYPOL <FREE) GOSF

ANALYSIS UNXTS LOW-LIMIT 2 HIGH-LiMfT -----w-w -w-w- --“------ we....-..----

lo-8 %

19.9 h

4.78 %

13.6 %

7.12 %

ZSL - PPM

1070, PPM

0.0110 % O.OLOO

. . THE LETTER CODE LOCATED BELOW EACH ASSAY IS A METKOD REFERENCE CODE, FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES l-8OCt-423-6832

- Ralston Purina Company Checkerboard Square - Saint Louis. MO 63164

_..

TO R.E,GOODMAN, CC K. S. PHILLIPS, ZRS

LAB NO 614493 ENTERkD 07/15/98 REPORTED 07/20/98

5713 M USD, GUINEA PIG DIET, ROOM TEMP. GROUP 8 MONSANTO R05G

ASSAY ..e---


PROT.COMB. (N X 6 -25) PRCB


FIBER (CRUDE) FIBR

ASH ASHF

I RON FEF

TOTAL VITAMIN C V-I-CA

GOSSYPOL (FREE) GOSF

ANALYSIS a.....-----

13.0

20.1

4.63 %

16.5

7.40

276.

754.

0.0360

UNITS LOWILIMIT 2 HIGH-LIMIT --w-w --s-..--- -...,---w--- %

%

%

%

PPM

PPM

% 0.100

THE LETTER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE- FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES l-800-423-6832

-

-

-

-

-

-

-

-

Ralston Purina Co~fip~~-:,~ Checkerboard Square Saint Louis. MQ 53 iiX

TO R.E.GOODMAN, CC K. S. PHILLIPS, 2RS

LAB NO 614695 EMEiED 07/15/98 REPORTED 07/30/98

5713 H REF, GUINEA PIG‘DIET, REFRKG, GROUP LO MONSANTO BBSG

ASSAY ANALYSIS UNITS -e--w w---e..-- we--- -w------- a-..--..----

MOISTURE AT ASSAY 7.72 % MOB

PROT.COMB.(N X 6.25) 20.4 % PRCB

FAT ( ETHER EXT. 1 4.79 YI FTEE

FIBER (CRUDE) 14.0 % FISR

ASH 7.68 % ASHF

I RON 275. PPM FEF

TOTAL VITAMIN C 1320s PPM VTCA

GOSSYPOL ( FREE) o.a100 % GOSF

CARBAMATE SCREEN C&P . CARBAMATE PESTICIDES

OXAMYL LESS THAN 0.05 PPM METHOMY L LESS 7HAN O-05 PPM 3-HYDROXYCARBOFURAN LESS THAN 0.05 PPM ALD I CARB LESS THAN 0 -05 PPM PROPOXUR LESS THAN 0.05 PPM CARBOFURAN CA&ARYL

LESS THAN a.05 PPM LESS THAN O-05 PPM

CARBANOLATE LESS THAN 0.05 PPM METHICC.4R3 LESS THAN 0.05 PPM

0,100

ORGANOPHOSPHATE PEST ORGP <PPM’: <PPM>

DI AZINON . . ..I...... LESS THAN 0.02 PARATHION.,,..,,,.. LESS THAN 0.02 DISULFOTON~......~. LESS THAN 0.02 THIMET e-.1......... LESS THAN O-02 ETHION....-........ LEtS,THAN 0.02 MIODAhJ . . . . . . . . . . . . LESS THAN 0.02 MALATHION . . . . . . . . . . LESS THAN 0.02 TFZIWION a.......... LESS THAN 0.02 METHYL PARATHION..- LESS THAN 0.02

Ralston Purina Company Checkerboard Square Saint Louis. MO 63164

TO R.E.GOODMAN, CC K. S. PHILLIPS. ZRS

-

-

LAB NO 614491 ENTERED 07/1!i/98 REPORTED 07/20/98

5713 K USD. GUINEA PIG DIET, ROOM TEMP. GROUP 6 MONSANTO BBSG

ASSAY --m-w

MOISTURE AT ASSAY Mea



FIBER (CRUDE) FIBR

ASH ASHF

I: RON FEF

TOTAL VITAMIN C VTCA

ANALYSIS --------

8.98

20.1

5.46

15.7

7.41

280.

1100.

UNITS LOW-LIMIT 2 HIGH-LIMIT ----- --..m.----- ..---,.a---^-

%

%

%

%

%

PPM

PPM

-

GOSSYPOL (FREE) GOSF

0.0370 % O.LOO

-

THE LETTER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE. FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES L-800-423-6832

-

-

-

-

-

-

Ralston Purina Company Checkerboard Square Saint Louis, MO 63764

&TO R.E.GOODMAN, ! ’

CC K. S. PHILLIPS, 2%. e%% p(-J#/y& *

:.A13 NO &+%96 ENTERE$ 07/15/98 REPORTED 07/30/98 0 ..e. , t 3,*&V.,

*>713 Z REF, GUINEA PIG DIET, REFRIG. GROUP 4 SANTO RBSG tbl-ngc/+-I &a- /bx

c - ” iSSAY ANALYSIS UNITS LOW-LIMIT 2 HIGH-LIMIT

----I ..M.m-“--- -m-w-

f”lOSSTURE AT ASSAY Ii

LO.4 % MOB

F’ROT.COMB. (N X 6 -251 s9.4 %

r” PRCB : ‘AT (ETHER EXT. 1 6.56 % b

FTEE FIBER (CRUDE) 13.6 %

FIBR 7.96 %

4SHF

TRON 280. PPM

9; FEF .

TOTAL VITAMIN C 1250. PPM

!- VTCA ;, bOSSYPOL (FR.EE) 0.0110 % in

GOSF p$ARBAMA?E SCREEN 0RBP

t ; CARBAMATE PESTICIDES * OXAMY L LESS THAN 0 -05 PPM

MEYHOMY L LESS THAN 0.05 PPM

7 3-HYDROXYCARBOFURAN LESS THAN 0.05 PPM

4-J ALDI CARB LESS THAN 0.05 PPM PROPOXUR LESS THAN 0.05 PPM

F CARBOFURAN LESS THAN 0.05 PPM

i ‘CAF&ARYL LESS THAN 0.05 RPM L,

CARBANOLATE LESS THAN 0.05 F’PM METHIOCARB

P LESS THAN 0 -0.5 PPM

----w-e-- -..--------

d@” ig 1.

, -4 5 -

q74i

0,100

L

C SRGAN~PH~SPHATE PEST ORGP (PPM) (PPM)

r DIAZXNON . . . . . . . . . . . LESS THAN 0.02 PARATHION.......... LESS THAN 0.02 F c 1 DISULFOTON......... LESS THAN 0.02 THIMET............. LESS THAN 0.02

EYHION . . . . . . ..I.... LESS THAN 0.02 MIODAN......,..... LESS THAN O-O2

k”; MALATHION.......... LESS THAN 0.02 TRIMION........... LESS THAN 0.02

i; METHYL PARATHION... LESS THAN 0.02

lerm “L?Ss Thnn” Is JSEO IO S49nly Ii%? !OWef llm of auamtatlon ct the ccc~u,e ““oc+~ (PR Conaitlc]nS emciov&. The “Se of the term “LBSS Thafl” dOeS nOl ImOly that

.‘XeS Ot JMlMFj WHe DreSf3Jt. %mOls SUOmNted 10 R&ton AnalyTicat LwratorlrjS for rOt~t,ne analysts WIII w retalna for a mmm’wtl Of thirty (30) days after Ihe report Of

j3naysls is ~SSue(l. Extenaed storage requirements must be brouqnt to tne artentlon of RalStoo Analyk!al Laboratones Onor to or at tne ttme 01 sample SUtWWSlOn.

Rll7lC-9008

‘i. Ralston Purina C0mp.w r Checkerboard Square Saint Louis. MO 63 164 _^._

PAGE 2 -

5713 H REF, GUINEA PIG DIET. REFRIG. GROUP la MONSANTO ,885G 5 # ,lPfkP

c+f. nv -L PESTICIDES & PC8 ... RSPS

(PPM) <PPM) ALDRIN . . ..-.--.--...LESS THAN 0.02 ENDRIN..,...,-.m..w LESS THAN 0.02 ALPHA-BHC .--...m-.- LESS THAN 0.02 HCB e .*.,.. s . . . . s-m-1. LESS THAN 0.02 BETA-BHC .-.--...-.. LESS THAN O-02 0 HEPTACHLOR..,.,..,. LESS THAN O-a2 DELTA-BHC.......... LESS 7HAN o-02 HEPTACHLOR EPOXIDE. LESS THAN a -02 CHLORDANE -w-w.----. LESS THAN 0.02 LINDANE,,...,s..,,. LESS THAN a.02 DDE .-.-..a......-.. LESS THAN a.02 METHOXYCHLOR.,..,.- LESS THAN a.02 DDT..CTOTAL).-....m LESS THAN 0.02 MIREX.,............ LESS THAN a.02 DIELDR1Nev.ee..,.,, LESS THAN a.02 PCB-,.....,....,e.. LESS THAN 0.15

-

-

-

-

THE LETTER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE. FCR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES l-800-623-6832

-

-

-

-

-

-

Ralston Purina Company Checkerboard Square Saint Louis, MO 63164

,

? :TO R _ Es GOODMAN,

ri

CC K. S. PHILLIPS, 2RS

~‘LAB No 624498 ENTERiD 07/15/98 REPORTED 07/30/98

5713 K REFt GUINEA'PIG DIET, REFRIG. GROUP 6 MONSANTO BB5G

k *

cI ASSAY ANALY SI S UNITS LOW-LIMIT 2 HIGH-LIMIT I ’ --c-w

i i MOISTURE AT ASSAY MOB

” PROT,COtlB, (N X 6.25% [I; PRCB ” FAT (ETHER EXT.)

‘c FIBEr::RUDE, ,I d FIBR

--“w--w- -w”-- -w------e e-------m-

11.6 %

ASH c D -i ASHF !a ” IRON

FEF ETOTAL VITAMIN C d : VTCA k GOSSYPOL (FREE)

I- GOSF & : CARBAMATE SCREEN

L9.6 %

S-66 %

16.5 %

T.77 %

259. PPM

1230, PPM

o,abra x

CR82

a.Laa

ia. -s CARf%AMATE PESTICIDES OXAMYL LESS THAN a.05 PPM

c METHOMYL % ,;

LESS THAN a-05 PPM 3-HYDROXYCARBOFURAN tfzss ~-IAN a -0% PPM ALD I C ARB LESS 7ki~N a.05 PPM

f PROPOXUR LESS THAN a .a5 PPM CARBOFURAN C&BARYL

LESS THAN 0.05 PPM ^

LESS THAN a.05 PPM

m CARBANOLATE LESS MAN 0.05 PPM

1 METHIOCARB LESS THAN 0.05 PPM bu

ORGANOPHOSPHATE PEST ORGP

E”1 (PPM) (PPM)

L 1 DIAZiNON..,,.,..... LESS THAN a.02 PARATHION.,....,.,. LESS THAN Q-02 DISULFOTON.~.~...~. LESS THAN a.02 MIME-T'............, LESS THAN Q-02

c EMION .-I---.---..- LESS THAN a.02 THIODAN.,.,.,..,,.. LESS THAN Q-02

ii. i MALATHION,,.,,,.,,, LESS THAN a.02 TRITHXON, . . . . .e -a .e LESS THAN 0.02 METHYL PARATHION.. - LESS 7lhAN a.02

- I.: ‘% tOrm “L?SS i%JbT’ IS USSO 10 S!pt’tiiy the lower IIfPlI of auant~tatt~n Of the OrmaUre Un@r the comj&onS emotOv& The USe Of Ihe term “Less Than” does not imory thn!

‘VXX%l OI jMlVlt? Were OfPjetlt. S~KWI~S SUbmRtSCJ 10 RafstOn Anal~caf LaQOrStOrwx for roUtme SnStySrS WM tx &na [Or a mttvmum Of thirty (30) (lays anw the rewn 01

3ntiySlS IS ISSJW.3. ~xtW’K%3d SlOwa rSClUbr9mentS must De bmugnt to the attention of R&ton AnalyUa &&OratorteS ~rlor to or at the rtme 01 Sample SUbmlSSlOn.

- Ralston P ufha Comparing Checkeboard Square! c Saint Louis, MO 63764 :

RT LAB NUMBER 614496 PAGE 2 -

5713 I REFr GUINEA PIG DIET. REFRIG, GROUP 4

MONSANTO BBSG

PESTICIDES & PCB -‘. RSPB

(PPM) - (PPM) ALDRIN-,,,,..,....,.LESS THAN 0.02 ENDRIN -----.I---.-. LESS THAN O-02 ALPHA-BHC,s...e.e-m LESS THAN O-02 HCB ~--~~-~1~--~-*.- LESS THAN O-02 BETA-BHC,.,e....,-, LESS THAN O-02 HEPTACHLOR-,. _._I.. LESS THAN 0.02 DELTA-Bf-lC,,...e-,-a LESS THAN 0.02 HEPTACHLOR EPOXIDE. LESS THAN O-02 CHLORDANE..-.--.-me LESS THAN 0.02 L1NDANE.ea-e-...... LESS THAN 0.02 DDE-...--.--.---..- LESS THAN 0.02 MFTHOXYCHLOR.,..... LESS THAN 0.02 DDT..(TOTAL)--.m... LESS THAN 0.02 M1REX.we.-w...,.,,, LESS THAN 0.02 DIELDRIN-.--...e... LESS THAN 0.02 PCB....,.....,,,.,, LESS THAN 0.15

THE LETTER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE.

FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES l-800-423-6832 -

Ralston Purina Comp3 !, Checkerboard Squan.

F

i 1

k ,TO R.E.GOODMAN.

1 LAB NO 614501 ENTERED 07/15/98 REPORTED 07/30/98

5713 M REF, GUINEA PIG DIET, REFRIG. GROUP 8 ’ kONSANT0 BB5G t I w .Y

ASSAY m -e--e r ; “MOISTURE AT ASSAY

MOB -PROT.COMB. (N X 6.25) i &.,

PRCB ‘FAT (ETHER EXT.‘)

FTEE FIBER <CRUDE)

%j FIBR ASH

m ASHF i, ]IRON

FEF -TOTAL VITAMIN C

E: VI-CA C-XISSYPOL (FREE>

GOSF ~?c~~RBAMATE SCREEN

ANALY SI S UNITS LOW-LIMIT 2 HXGH-LIMIT -----w-w --m-e m----w--- -----e----

12.-z %

13.8 %

5.44 %

17.8 %

8.08 %

280. PPM

1220. PPM

0.0400 3:

CRBP

a.100

e: CARBAflATE PESTICIDES OXAMYL LESS THAN 0.05 PPM

ri METHOMY L LESS THAN 0.05 PPM

t i 3-HYDROXYCARBOFURAN LESS THAN 0.05 PPM ALDICARB LESS THAN 0.05 PPM

r PROPOXUR LESS THAN 0.05 PPM CARBOF’JRAN LESS THAN 0.05 PPM

. 1 CARBARYL LESS THAN 0.05 PPM CARBANQLATE

i? LESS THAN 0.05 PPM

ti + MElHIO.:ARB LESS THAN 0.05 PPM Ls _I

c.!?GANOPHOSPHATE PEST ORGP (PPM) (PPM)

DIATXNON........... LESS THAN 0.02 PARATHION.......... LESS THAN 0.02 P, _1 DISULFCTON......... LESS THAN 0.02 THIMET............. LESS THAN O-02

c ETHION............. LESS THAN 0.02 THIODAN............ LESS THAN 3.03 MALATHiON.......... 0.10 TFZITHEON........... LESS THAN C-02

6 I METHYL PARATHION.. . LESS THAN 0.02

[; ; ‘C El!7 “t?SS Than” IS USed (0 SlC~nl&’ fne Io#w llmlr 01 ouaritlratlon 01 me crocwure unaer lne ~OncjltlonS SmOlovw. The uSe of the term ‘T-$SS Than” does oat lmalv In:’

raCL?S 01 anaIVE? Were prtsefll. SamDkS SuOmlned lo hISOn Analyu~t LJOO~XO~S 1or routme analvS!S wl~( m retalna iOr a mmlmum Or tMIy (30) days aner ltle rewn c.!

InalySlS IS ISSUeQ. Exteflaed Storage r~U:remen!S muS1 be brouqnt to lne SttentgOn Of fQ[StOn Analytical WratOrleS pnor ln Or af tJ?e tm-te Ol Sample sub4lXSSlon.

“....I_ nn,,

- f$ftY7t&

RT LAB NUMBER 6 14438

Ralston Purina Company r, Checkert3oard Square Saint Louis, MO 63164

TX- PAGE 2

MONSANTO BBSG

PESTICIDES & PCB .‘. RSPB (PPM)

ALDRIN . ..w-..a..... LESS THAN 0.02 ALPHA-BHC.,..,..... LESS THAN 0.02 BETA-BHC.,..,...... LESS THAN 0.02 DELTA-BHC.,,..,.,., LESS TWAN 0.02 CHLORDANE-......... LESS THAN 0.02 DDE e....-.-e....... LESS THAN 0.02 DDT..(TOTAL),.,..,, LESS THAN 0.02 DIELDRIN .rr.rrr...r LESS THAN 0.02

5713 K REF, GUINEA PIG DIET. REFRIG. GROUP 6

ENDRIN . . . ..w.-..... LESS THAN 0.02 HCB,.......,,....., LESS THAN 0.02 HEPfACHL0R.m....... LESS THAN 0.02 HEPTACHLOR EPOXIDE. LESS THAN 0.02 LINDANE....,....,,. LESS THAN 0.02 METHOXYCHLOR..,...s LESS THAN Q-02 MIREX....s.....,.,. LESS THAN O-02 PCB,........,...,., LESS THAN 0.15

THE LETTER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE. FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES l-800-623-6832

-

-

-


Monsanto Company MSL-15763 Biotech Regulatory Sciences Page #: X85 of 197

7.5 Feed Analysis Post-Day 21

a

c /

-

PAGE 2 -

5713 M REF, GUINEA PIG DIET, REFRIG, GROUP 8 MONSANTO BBSG

PESTICIDES & PC8

ALDRIN ............. ALPHA-BHC .......... BETA-BHC ........... DELTA-BHC .......... CHLORDANE .......... DDE ................ DDT .. (TOTAL] ....... DIELDRIN ...........

RSPB (PPM) LESS THAN 0.02 LESS THAN O-02 LESS THAN 0.02 LESS THAN 0.02 LEss 7-m~ o-at LESS THAN 0.02 LESS THAN 0 -02 LESS THAN 0.02

- ENDRIN ............. HCB ........ .r..*- ... HEPTACHLCJR ......... HEPTACHLOR EPOXIDE.

LINDANE ............ HETHOXYCHLOR ....... MXREX .............. PC8 ................

LESS THAN 0.02 - LESS THAN O.&Z LESS THAN 0 -02 LESS THAN 0.02 LEss ~-IAN 0.02 LESS THAN 0.02 LESS THAN 0.02 LESS THAN 0.15

THE LETTER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE. FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES 1-800-623-6832

-

-

-

-

-

-

f,:TO R. GOODMAN. CC K. S. PHILLIPS, .ZRS

$: LAB NO 6lYri50 ENTERED’98/18/98 REPORTED 10/22/98

: 1 GUINEA PrG 9ONTROL DIET 5713C-H BOX 1 V98.003.3006 LCJT9%8-I

a- MONSANTO t ,i

’ ASSAY ANALYSIS UNITS LOW-LIMIT 2 HIGH-LIMIT

fy ----- -w-e--..- --e-w ------.w-- --w-------

i :TOTAL VITAMIN C 1160. PPM VTCA

CONTRACT ANALYSES AFLATOXIN 81

OUT LESS WAN 1.0 PPS

r : Bt GL

k” G2 ; : AFLATOXIN PERFORMED i /

OCHRATOXL N A

r CITRININ

k 2 TRXCHOTHECENES b? 73 T-Z TOXIN

M-2 TOXIN

; DIACETOXYSCIRPENOL NEOSOLANIOL FUSARENON-X DEOXYNIVALENOL NIVALENOL ZEARALENONE

p FUMONISIN B1 FUMONI SIN B2

6 : FUMONI SIN 83 Z 5 ACETYL-DON

3 ACETYL-DON

LESS THAN 1.0 PPB LESS THAN 1.0 PPB LESS THAN 1.0 PPB BY HPLC METHOD LESS THAN 5 PPB LESS THAN 0.2 PPM

LESS THAN 0.1 ‘PPM LESS THAN 0.1 PPM LESS THAN 0.3 PPM LESS THAN 0.5 PPM LESS THAN 0.5 PPM

0.2 PPM LESS THAN 0.5 PPM LESS THAN LOO PPB LESS THAN 0.1 PPM LESS THAN 0.1 PPM LESS THAN 0.1 PPM LESS THAN a.1 FPM LESS THAN 0.1 PPM

THE LETTER CODE LOCATED BELOW EAC:t ASSAY IS A METHOD REFERENCE CODE. FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES l-800-423-6832

F The term “Less TharP IS used to slgnlfy the lower llmlt of auantitaston of the prwadure under tne conditions empfoyed. The use 01 the term “Less Than” does nut KWIV :I

:racf% cl anaivte were preSent. SJmDteS suomltled to Ralston Analytcal Laboratone for rouane analystswill be retalnea for a mmtmum of thtny r3G) &ys aner ‘he ic::: Jnalvsts IS issued. Extenaed storage rec$UliemenIS must Da brougnt lo the anenllon of RalsWJn Analytical Laboiatorles onor lo or ar ine time OI samole suom!ss\on

nt,7*.

TO R. GOODMAN, CC K, S. PHILLIPS, ZRS

LAB NO 619453 ENTERED OS/18798 REPORTED LO/22/98

Ralston Purina Company Checketioard Square Saint Louis, MO 63164 ._.I.,.

-

-

MOD FUINEA PIG W/10% CS-A 5713C-I BOX 1 S98.OL9.3044 LOT9848-2 MONSANTO

ASSAY ANALYSIS UNITS --w-w --C.-m.--- e--m-

TOTAL VITAMIN C 1440, PPM VTCA

CONTRACT ANALYSES OUT AFLATOXIN BI LESS THAN 1.0 PPB AFLATOXIN 82 LESS THAN 1.0 PPB AFLATOXIN Gl LESS THAN L-0 PPB AFLATOXIN G2 LESS THAN 1 .O PPB OCHRATOXIN A LESS THAN 5-O PPB CITRININ LESS THAN O-2 PPM T-2” TOXIN LESS THAN 0.1 PPM HT-2 TOXIN LESS THAN 0.1 PPM DIACETOXYSCIRPENOL LESS THAN O-3 PPM NEOSOLANI OL LESS THAN 0.5 PPM FU SARENON X LESS THAN 0.5 PPM DEOXYNIVALENOL 0.2 PPM 15 ACETYL-DON LESS THAN 0.1 PPM 3 AC ETY L-DON LESS THAN 0.1 PPM NIVALENOL LESS THAN 0.5 PPM ZEARALENONE LESS THAN 100 PPB FUMONf SIN 81 LESS THAN 0.1 PPM FUMONI SIN 82 LESS THAN 0.1 PPti FUMONISIN 83 * LESS THAN 0.1 PPM

LOW-LIMIT 2 HIGH-LIMIT -m------- ..----^---..

-

THE LET-TER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE. FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES l-800-423-6832

-

-

-

Ihe term ‘-Less Than” IS used lo stgmly the lower llrnlt of quant!tatlon of lhe procedure under tne condllions emoioyea. ihe use of the term “Less Than” r!oes not imply that

:rxes ol dnalvxe were cresem. Samples submtk?d to Ralston Analytical LaWratorles tar rouvne anaiysls ~111 De re!alnM for a mnmum 01 thvty (30) days alter rhe repon of

;toalysrs IS ISSUW. Extenaea storage requirements must be orougnt to the attention of Aalsron AnalyWal LaboratorIes trier to cr at the tlme 01 sample SubmSStOn.

R1~71CSOOE

Rakton Purina Company cb8Ck8fbOSd SqUare Saint Louis, MO 63164

TO R. GOODMAN. CC K. S. PHILLIPS, 2RS

LA8 NO 619452 ENhRED 08/18/98 REPORTED LO/22198

GUXNEA PIG CONTROL DIET 5713C-H BOX 3 V98.003.3004 tOT9848-1 MONSANTO

ASSAY ANALYSIS UNITS -e-w- --e----- s--m- TOTAL VITAMIN C 1580. PPM

VTCA CONTRACT ANALYSES OUT

AFLATOXIN Bi LESS THAN 1.0 PFa 02 LESS THAN 1.0 PPB Gi LESS THAN 1.0 PPB Gt LESS THAN 1 .O PPB

AFLATOXIN PERFORMED BY HPLC METHOD OCHRATOXIN A LESS THAN 5 PPB CITRININ LESS THAN 0.2 PPM

TRICHOTHECENES T-2 TOXIN LESS THAN 0.1 PPM HP-2 TOXIN LESS THAN 0.1 PPM DIACETOXYSCIRPENOL LESS THAN 0.3 PPM NEOSOLANIOL LESS THAN 0.5 PPM FUSARENON-X LESS THAN 0.5 PPM DEOXYNIVALENOL 0.2 PPM NKVALENOL LESS THAN 0.5 PPM ZEARALENONE LESS THAN 100 PPB FUMONI SIN 61 LESS THAN 0.1 PPM FUMONI SIN 82 LESS THAN 0.1 PPM, FUMONISIN 83 LESS THAN 0.1 PPM 15 ACETYL-DON LESS THAN 0.1 PPM

3 ACETY L-DON LESS THAN 0.1 PPM ,.. ;,\

LOW-LIMIT Z HIGH-LIMIT ----..-..-- ---------..


fhe term “Less Than’ is used to scgmfy Ihe lower timlt of quantltabon of the orocedure under the conaitions emoroyea. The use 01 Ihe term “Less Than” does nor imoly that :races of analve were present. Sam&s submtned to Ralston Analyrvzal Lxxxatones for routme analysts WIII be retaxxzo Ior a mm!mum of thlfty (30) days atter the rerxn of .lnalys!s IS issueci. Exlenoed sforaqe requtrements mlJst be Drouqnt to Ih8 anenhon of Rafslon Analytical Laborator% trlor to or al the Ilme Of Sampk? sucmlsslon.

RI1710 9COO

Ralston Purina Cornpar, Checkett2oard Square Saint Louis, iU9 63164 -.

TO R. GOODMAN, CC K. S. PHXLLIPS, 2RS

LAB NC 619454 ENTERED 081l.8198 REPORTED 10/22/98

MOD GUINEA PIG W10% CS-A 5713C-I BOX 2 S98.019.3064 LOT9848-2 MONSANTO

ASSAY ANALY SI S UNITS -......e -------a --w-w TOTAL VITAMIN C 1550, PPM

VTCA CONTRACT ANAtYSES OUT

AFLATOXIN BL LESS THAN 1 .O PPB

AFLATOXIN B2 LESS THAN 1.0 PPB AFLATOXIN Gl LESS THAN 1-O PPB AFLATOXIN G2 LESS THAN 1.0 PPB OCHRATOXIN A LESS THAN 5-O PPB CITRININ LESS THAN 0.2 PPM T-2 TOXIN LESS THAN 0.1 PPM HT-2 TOXIN LESS THAN 0.1 PPM DIACETOXYSCIRPENOL LESS THAN 0.3 PPM NEOSOLANIOL LESS THAN 0.5 PPM FUSARENON X LESS THAN O-5 PPM DEOXY,NIVALENOL 0.2 PPtl 15 ACETYL-DON LESS THAN 0.1 PPM 3 ACETY L-DON LESS THAN 0.1 PPM NIVALENOL LESS THAN O-5 PPM ZEARALENONE LESS MAN 100 PPB FUMONI SIN Bl LESS MAN 0.1 PPM FUMQNISIN 82 LESS THAN 0.1 PPM FUMONI SIN 83 LESS THAN 0.1 PPM ”

LOW-LIMIT 2 HIGH-LZMIT ---..m.-.m-I N----M.")---

-


The term .‘Less Than” IS use0 lo SlgnlfV the lower Ilm#f of quantitation of the procedure under the conaitlons emptcyed. The use of lhe term ‘LPSS Than” does n0l imo?: “’

:rxcs of anatvte were present. Samples suDmmed IO Ralston AnalytIcal Laoora!ones for rautrne analysts w111 t?e retalnw for a mmlmum of Ihinv t3r?) Cays arter the re~?c’

analysts IS issueo. Exlenaed storage requirements must be Drouqnt to the anention of Ralston Anaiytical Labcrararres cnor to or at the !rme of samore SuDm!SS~on.

Rl171G.,

Ralston Purina Company Checkerboard Sqwua Saint Louis, MO 631&s ---^_.

VT0 R. GOODMAN,

ki

CC K. S. PHILLIPS, ZRS

mLAB NO 619456 ENTERED 08/18/98 REPORTED 10/22/98 I i - : f: ‘MOD GUINEA PYG’W/LO% CS-8 5713C-K BOX I S98.023.3044 LOT9848-4

MONSANTO

ANALYSIS UNITS .,--we ----..--- ..-..--

FTOTAL VITAMIN C 1150. PPM i ) VTCA L Id

CONTRACT ANALYSES OUT

r

AFLATOXIN Bl LESS THAN I .O PPB AFLATOXIN 82 LESS THAN 1.0 PPB AFLATOXIN Gl LESS THAN 1.0 PPB AFLATOXIN Gt LESS THAN 1.0 PPB

p OCHRATOXIN A LESS THAN 5.0 PFB

c ,, CITRXNIN LESS THAN 0.2 PPM T-2 TOXIN LESS THAN 0-r PPM

I? HT-2 TOXIN LESS THAN 0.1 PPM

I L DIACETOXYSCIRPENOL LESS .I%iAN 0.3 PPM NEOSOLANLOL LESS THAN 0.5 PPM

c

FLISARENON X LESS THAN 0.5 PPM DEOXYNIVALENOL a.3 PPM

i i 15 ACETY L-DON LESS MAN 0.1 PPM 3 ACETYL-DON LESS THAN 0.1 PPM

y-‘ NIVALENOL LESS THAN 0.5 PPM

b ZEARALENONE LESS MAN LOU PPB FUMONX SIN 81 LESS THAN 0.1 PPM

I- FUMONI SIN 82 LESS THAN 0.1 PPM ti ’ FUMONISIN 83 LESS THAN 0.1 PPM L d

LOW-LIMIT 2 HIGH-LIMIT ..-------- ------..---

C’rHE LETTER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE. i .s r FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES l-800-423-6832

The term “Less Than‘ IS used to slgnlfy the lower tImIt 01 ouanutatlon of the procedure under the con&ions emoloyed. The use of Ihe term “Less Than” does nor Imply rhnc

p Jxes of analvte were preseor. SampieS subm!ned to Ralston AnalytIcat LaCoratones lor routme analysis V/III tx retalnea !cr a mmimum o? ;hir’Q (30) days a&r tne ret%- 0’

t : ,znalWs IS %sueo. Extenoeu storage raqu~rements must be orouqnt to the anenhon of Ralston Analytxal Laborarortes cnor to or at the time or sample suom6.slon.

E:il?lC-"Oill - II

Ralston Purina Company Checkerboad Square Saint Louis, MO 63164

TO R. GOODMAN, cc K, S. PHILLIPS, 2RS

LAB NO 619458 .” -

ENTERED 08/18/98 REPORTED 10/22/98

MOD GUINEA PIG W/LO%CS*B BOX 3 5713C-K 898.021.3044 LOT9648-4 MONSANTO

LOW-LIMIT Z HIGH-LIMIT --------- -----w----

97

ASSAY ANALY SI S UNITS ----- -----em.. ..---...

TOTAL VITAMIN C 1250, PPM VTCA

CONTRACT ANALYSES OUT AFLATOXIN Bl LESS THAN 1 .O PPB AFLATOXIN 82 LESS THAN 1 .O PPB AFLATOXIN Gl LESS THAN 1 .O PPB AFLATOXIN Gi! LESS THAN 1.0 PPB OCHRATOXIN A LESS THAN 5-O PPB CITRININ LESS THAN O-2 PPM T-2 TOXIN LESS THAN 0.1 PPM HT-2 TOXIN LESS THAN 0.1 PPM DIACETOXYSCIRPENOL LESS THAN o-3 PPM NEOSOLANIOL LESS THAN 0.5 PPM FUSARENON X LESS THAN O-5 PPM DEOXYNIVALENOL O-3 PPM 15 ACETYL-DON LESS THAN 0.1 PPM 3 ACETYL-DON LESS THAN 0.1 PPM NIVALENOL LESS THAN Ct.5 PPM ZEARALENONE LESS THAN 100 PPB FUMONISIN 81 LESS THAN 0.1 PPM FUMONI SIN B2 LESS THAN 0.1 PPM FUMONI SIN B3 LESS THAN O-1 PPM

-

-

-

-

-

-e

-

-

THE L’ETTER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE- FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES L-800-423-6832

fha term “Lt?ss Than” IS used to signtfy the lower ltmtt ot cuantltatlon of the procedure under tne canallions emaloyed. The use of the term “LBSS Than” does not rmow mat

:mces of anaryte were present. Samples submmed to Ralston Analyvzaf Laooraronss lor rouone analysis wit be reiavwa 101 a minrmum of rhlr?y (30) days after Ine rexn ii:

.X~aIVSIS is ISSUM. Ex!onced Storage requlremenls must be orougnt lo the anentron of Rals!on AnalyWal LabOrStOrIeS CrlOr t0 Or at the itme Ot Samp@ SUtXIXSS~On.

Rll7!SfC08

Ralston Purina Coi:~p~ :, Checkerboard Squ.w: Saint Louis, MO C;I :::,. _---.-

F t ”

r?

TO R. GOODMAN. cc K. S. PHILLIPS, 2RS

,g \\l2o 4 b \

LAB NO 619457 ENTERED a/18/98 REPORTED 10/22/98 6

MOD,GUXNEA PIG W/10% CS-I3 5713C-K BOX 2 SY8.021.3046 LOT. 9848-4 MONSANiO

ASSAY ANALYSIS UNITS -e....- ------w.- e-d-- TOTAL VITAMIN C 1160. PPM


AFLATOXIN 81 LESS THAN 1.0 PPB AFLATOXIN Bt LESS THAN 1 .O PPB AFtATOXIN Gl LESS THAN 1.0 PPB AFLATOXIN G2 LESS THAN 1.0 PPB OCHRATOXIN A LESS THAN 5.0 PPB CITRININ LESS. THAN 0.2 PPM T-2 TOXIN LESS THAN 0.1 PPM H-l--Z TOXIN LESS THAN 0.1 PPM DIACETOXYSCIRF'ENQL LESS THAN 0.3 PPM NEOSOLANIOL LESS THAN 0.5 PPM FUSARENON X LESS THAN 0.5 PPM DEOXYNIVALENOL 0.3 PPM 15 ACETYL-DON LESS THAN 0.1 PPM 3 ACENL-DON LESS THAN 0.1 PPM NIVALENOL LESS THAN 0.5 PPM ZEARALENONE LESS THAN 1OC PPB FUMONISIN 81 LESS THAN 0.1 PPM FUMONI SIN BZ LESS THAN 0.1 PPM FUMONISIN 83 LESS THAN 0.1 PPM

LOW-LIMIT 2! .HIGH-LIMIT M--w--..-- ..--.....-----

THE LETTER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE.

* d FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES 1-800-423-6832

r :

The Iafm “LOSS Than’ IS USed tQ Slgnlfy lhe !OWef llmrl of cuanttration ot Iha procedure under lna CondltlonS amoioyad. The use 01 the tarm “LeSS Than” does no1 lmnty th;~

l-7 :r3CaS Ot dnatsw were W-Sent. S3mDtaS SubmUted to RatsIOn AnalVwal LaDoratorlaS for curme analvs~s wtit M retalnea for a mmlmum of lhlrtv (30) days Mar WI TE’WCI (;:

%1

3natVslS IS NJW. Emwed Storage WulEmenlS must be orouqnt !o rhe anention 01 Ralston Anatykxl Laooratorres srlor IO or al tne tcme ot sample suomtssion.

R 1171 C;-SiY!l1

TO R. GOODMAN, CC K. S, PHILLIPS, ZRS

7

LAB NO 619455 ENTERED OS/L8198 REPORTED 1’0/22/98 p\ \s

fj 2o a

MOD GUINEA PIG W/laXCS-A 5713C-I BOX 3 S98,039,3C44 LOT9848-2 MONSANTO -

ASSAY ANALY SI S UNITS --a-- -------- w--e- TOTAL VITAMIN C 1460. PPM


AFLATOXIN Bl LESS THAN 1.0 PPB AFLATOXIN Bt LESS THAN 1.0 PPB AFLATOXIN Gl LESS MAN 1.0 PPB AFLATOXIN G2 LESS THAN 1.0 PPB OCHRATOXXN A LESS THAN 5.0 PPB CITRININ LESS THAN 0.2 PPM T-2 TOXIN LESS THAN 0.1 PPM HT-2 TOXIN LESS THAN 0-L PPM DKACETOXYSCIRPENCL LESS THAN 0.3 PPM NEOSOLANIOL LEss THAN a.5 PPM FUSARENON X LEss THAN a.5 PPM DEOXYNIVALENOL a.2 PPM 15 ACETYL-DON LESS THAN a.1 PPM 3 ACETYL-DON LESS THAN 0.1 PPM NIVALENOL LESS .THAN a.5 PPM ZEARALENONE LEss ~-HAN 100 PPB FUMONISIN 81 LESS THAN a.1 PPM FUMONI SIN 82 LESS THAN a.1 PPM FUMONISIN 83 LESS THAN a.1 PPM

LOW-LIMIT 2 HIGH-LI!ttfT

.

‘- THE LE-JTER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE. FOR AibJITIONAL INFORMATION CONTACT CLIENT SERVICES l-800-423~6,832

.

The term “Less TharV is USed IO stgnlfy the lower llmtl of quan!tlatron of {he procedure under rbe uvldilions emolcyed. The use of :he term “Less Than” &es 13~1 if~‘@y Inat !raCes of analvte were presenl. SXIIO~~S submWd lo Ralston Analylcal LaCwxarorls for ro!mne anal.lysIs will be relamed for a mlnimum or thtrly (J()) days a&f t&e tef&,r~f

_

;InaIvSS is 15suHl. Extenocd srorage reQulremenfs must be brougnt to ihe altenkon of Rats&n AnalytCal Laboralones cnor IO or at the t!me 01 sample suom!sswn.

A1171Q4~308

F R. GOODMAN,

Ld

Ralston Purina Company Checkerboanl Square Saint Louis, MO 63164

CC K. S. PHILLIPSt ZRS

*AB No 619460 ENTERED a8/18/98 REPORTED la/22198 t-j MOD GUiNEA PIG H/la% CS.-D 5713C-M BOX 2 598.023.3044 LOT9868-6

NSANTO

m&SAY ANALYSIS UNITS LOW-LIMIT 2! HIGH-LIMIT --v-w --m-w--- -e--w ..-..---s-- ----------

7CTAL VITAMIN C 1110. PPM t : VTCA CCNTRACT ANALYSES OUT

p AFLATOXIN Br LESS THAN 1.0 PPB AFLATOXIN 52 LESS THAN 1 .a PPB

b- , AFLATOXIN Gl LESS THAN 1 .O PPB

h”r AFLATOXIN G2 LESS THAN I .O PPB

b , OCHRATOXI N A tEss THAN 5. a pp0

h, CITRININ LESS THAN 0.2 PPM T-Z TOXIN LESS THAN a.1 PPM

r t-IT-2 TOXIN LESS THAN 0.1 PPM

b-9 DIACETOXYSCIRPENOL LESS THAN a.3 PPM NEOSOLANIOL LESS THAN 0.5 PPM FUSARENON X LESS THAN 0.5 PPM

k : DEOXYNIVALENOL a.3 PPM Cd

15 ACETYL-DON LESS THAN 0.1 PPM

6*1 3 ACETYL-DON LESS THAN a.1 PPM t 1 NIVALENOL !

LESS THAN 0.5 PPM b ZEARALENONE LEss WAN la0 PPB

FUMONI SIN Sl LESS -THAN 0.1 PPM *F” FUMONI SIN 82 LESS THAN a.1 PPM (i FUMONI SIN 83 L j LESS THAN a.1 PPM

: THE LE;f”TER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE. ?OR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES l-800-423-6832

-’ e term “Less Than’ !s used lo signify the lower Ilmtf of cuantttatlon of the procedure under the conditions emoloyeo. The use oi the term “Less Than” does noI imoly that ’ .ces 01 anatyte were present. Samples submmed IO Ralston ~nalyrrca~ LJwratones for routme analysts WIII be re!alnea !of a mmimum Of lnmv GO) days alter tne repor? of i. jatvsis 6 ISSUM. Ex:enaed storage requ!rements must ce brwqnt to the anentton of Ralston Analyticz9 Laooratones orlor to or at the ttme Ot Sample SubmlsslOn.

.cl,,-r,P QrTil(l

TO R. GOODMAN. CC K. S. PHILLIPS, 2RS

LAB NO 619459 EN%ERER,08/18/98 REPORTER 10/22/98

MOD GUINEA PIG WILOr. CS-D 5713C-M BOX 1 S98.023.3044 LOT9848-6 MONSANTO

LOW-LIMIT i! HIGH-LIMIT ------m-v --...-w----I

o-2-

ASSAY ANALY SI S UNITS --w-- --B-m...-- ----- TOTAL VITAMIN C 793. PPM


AFLATOXIN Bl LESS THAN 1.0 wa AFLATOXIN BZ LESS THAN 1 .O PPB AFLATOXIN Gl LESS THAN 1.0 PPB AFLATOXIN G2 LESS THAN 1.0 PP8 OCHRATOXIN A LESS THAN 5.0 PPB CITRININ ixss THAN a.2 PPM T-Z TOXIN LESS THAN 0.1 PPM HT-2 TOXIN LESS THAN 0.1 PPM DIACETOXYSCIRPENOL LESS THAN 0.3 PPM NEOSOLANIOL LESS THAN 0.5 PPM FUSARENON X LESS THAN 0.5 PPM DEOXYNIVALENOL 0.3 PPM 15 ACENL-DON LESS THAN 0.1 PPM 3 ACETYL-DON LESS THAN 0.1 PPM NIVALENOL LESS THAN 0.5 PPM ZEARALENONE LESS THAN 100 PPB FUMONI SIN Bl LESS THAN 0.1 PPM FUMONISIN 82 LESS THAN 0.1 PPM FUMONX SIN 83 LESS THAN 0.1 PPM

-

-

-

-

-

-

THE LEmER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE. FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES 1 -ma-423-6832

7% Ierm “LOSS Than :s used to $rgnrfy the tower llmlt of ouantfration of the oroc~ure under the conctrlions emoicyed. The use ol the Iefm “Less Than” does r.01 ~moly !;I I! :rxcs 01 anawe were Dresent. Samples submrned to Ralston Ana:ytcal Laaoraronw for routine anatysls W&II he ratawxl lor a mtnlmum 01 ttwty (30) days attar the reX3C:i csi analysts IS CWIXY. Extenaea storage requlraments must be brougnl to tne aRent!on 01 Ralston Cna~ytrcat Laboratones onor to cr ar the tlme ot sample sucrnlss~on.

RlIy!(1-&;!“~

TO R. GOODMAN. CC K. S. PHILLIPS, tRS

LAB NO 619462 ENTERED 08/18/98 REPORTED lO/LZ/SH

$pj \\\Jq c6

0 ,A’

3d

’ f p-b ,, >

MOD GUINEA PIG W/lO%CS-D 5713C-M BOX 3 s93.023.3044 LOTy843-6 4

MONSANTO fa-ct’

s&S

ASSAY ANALYST S UNITS LOW-LIMIT Z HIGH-LIMIT -v--e --I---..- e-L-- “---....-..- -----e..--..

TOTAL VITAMIN C 1090. PPM VTCA

CONTRACT ANALYSES OUT AFLATOXIN 51 LESS THAN 1 .O PPB AFLATOXXN BZ LESS THAN L-0 PP8 AFCATOXfN GL LESS THAN I .O PPB AFLATOXIN G2 LESS THAN Z -0 PPB OCHR ATOX I N A LESS THAN 5.0 PPB

CXTRXNIN LESS THAN O-2 PPM

T-Z TOXIN LESS THAN U-1 PPM HT-2 TOXIN LESS THAN 0. I Pf=M

DIACETOXYSCIRPENOL LESS THAN 0.3 PPM NEOSOLANIOL LESS THAN 0.5 PPM FUSARENON X LESS THAN 0.5 PPM DEOXYNIVALENOL 0.3 PPM

15 ACETYL-DON LESS THAN 0.1 PPH 3 ACETYL-DON LESS THAN 0.1 PPM NI VALENOL LESS THAN 0.5 PPM ZEARALENONE LESS THAN 100 PPB FUMONI SIN Bl LESS THAN 0.1 PPM FUMONI SIN 82 LESS THAN 0. L PPM FUMONI SIN 83 LESS THAN 0. I PPM

THE LETTER CODE LOCATED BELOW EACH ASSAY IS A METHOD REFERENCE CODE- FOR ADDITIONAL INFORMATION CONTACT CLIENT SERVICES I.-800-423-6832

‘he term “Less Than” IS used IO signify lhe lower ltmil of ouan[ltatton of the procedure under tne ~nd~l~ons emcroyso The use ol the tern1 “t-ass Than” &es not W:WJ Ii I$: :ixes cl analwe were present. S amPi= submWd IO Ralston Analytical Cabofarones tar ~C?JKW iinatysts G~~~l ce re!aw~ !or a minimum 3r :nicty [?O) Gays nner tne IGCW CI

:naws 1s w@. Exwoed storage rwuremms must be brougnt to the affentlon 01 Ea!sron Anayricar Labora:orles prior 10 or at me lime ot sample suwn~s~~on

R, t 7: :;- :c:,:;

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